Compositions, methods, systems and kits for nucleic acid amplification and analysis

ABSTRACT

The disclosure provides compositions, methods, systems and kits for amplifying and assaying nucleic acids.

CROSS-REFERENCE

This application is a continuation of PCT International Application No. PCT/US2015/061252, filed Nov. 18, 2015, which claims priority to U.S. Provisional Patent Application No. 62/082,534 filed on Nov. 20, 2014, U.S. Provisional Patent Application No. 62/082,538 filed on Nov. 20, 2014 and U.S. Provisional Patent Application No. 62/082,541 filed on Nov. 20, 2014, which applications are herein incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 30, 2017, is named 46846701301SL.txt and is 757 bytes in size.

BACKGROUND

Nucleic acid amplification methods permit amplification of nucleic acid molecules in a sample, such as a biological sample. A nucleic acid molecule can be amplified, via, for example, thermal cycling based approaches (e.g., polymerase chain reaction (PCR)) or via isothermal approaches. Following amplification of a nucleic acid molecule, amplification products can be detected and the results of detection interpreted by an end-user. Nucleic acid amplification may also be useful in preparing a nucleic acid molecule for subsequent analysis in numerous applications related to nucleic acid analysis such as, for example in detecting target nucleic acid sequences, single nucleotide polymorphisms (SNPs), sequence mutations (e.g., deletions, insertions), detecting rare nucleic acid molecules/sequences in a sample and/or preparing a nucleic acid molecule for a sequencing reaction. Thus, due to the applicability of nucleic acid amplification to a wide range of applications, there exists a need for compositions, kits, methods and systems useful for amplifying nucleic acid molecules and/or for analyzing amplified nucleic acid molecules that are generated from nucleic acid amplification.

SUMMARY

The disclosure provides compositions, kits, methods and systems for the amplification and analysis of nucleic acids. In addition, the disclosure provides compositions, kits, methods and system for the generation and analysis of complexes that comprise particles and nucleic acids. The methods and compositions disclosed herein find a wide array of utilities in, for example, research, environmental testing, forensic identification and clinical diagnostics.

An aspect of the disclosure provides a set of particles for nucleic acid amplification. The set of particles can comprise a first particle that comprises a first primer having a 5′ end and a 3′ end, where the first primer is linked to the first particle via the 5′ end of the first primer, and where the first primer exhibits sequence homology to a target nucleic acid strand at a 5′ end of the target nucleic acid strand. The set of particles can also comprise a second particle comprising a second primer having a 5′ end and a 3′ end, where the second primer is linked to the second particle via the 5′ end of the second primer, and where the second primer exhibits sequence homology to a complement nucleic acid strand of the target nucleic acid strand at a 5′ end of the complement nucleic acid strand. Moreover, a sequence on the target nucleic acid strand to which the first primer exhibits homology can be different than a sequence on the complement nucleic acid strand to which the second primer exhibits homology.

In some embodiments, the first particle and/or the second particle can be contained in a partition that can be, for example, a droplet in an emulsion, a well or a vessel. In cases where the partition is a well, the well can be well among an array of wells. In some embodiments, the 3′ end of the first primer may be adapted to be extended in a primer extension reaction to form a copy of the target nucleic acid strand or a portion thereof. In some embodiments, the 3′ end of the second primer may be adapted to be extended in a primer extension reaction to form the complement nucleic acid strand or a portion thereof. In some embodiments, the first particle may comprise at least two of the first primer and/or the second particle may comprise at least two of the second primer. In some embodiments, the set of particles can further comprise a third particle that may comprise the first primer and/or second primer.

In some embodiments, the first and/or second particle may be selected from the group consisting of solid particles, porous particles, nanoparticles, beads, microparticles, metal particles, magnetic particles, semiconductor particles, polymeric particles, and nucleic acid particles. In some embodiments, the first or second primer may be directly linked to a respective one of the first and second particles. In some embodiments, the first or second primer may be linked to a respective one of the first and second particles through at least one linker. The linker can be selected from the group consisting of a nucleic acid, a phosphate moiety, an amino acid, a peptide, a hydrocarbon chain, a polyethylene glycol (PEG), a polysaccharide and combinations thereof. In some embodiments, the first and/or second particle may comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof. In some embodiments, the first particle and/or second particle may have a dimension of about 0.5 nm to about 100 nm. In some embodiments, the first particle and/or second particle may have a dimension of about 1 nm to about 20 nm.

An additional aspect of the disclosure provides a set of particles for nucleic acid amplification. The set of particles can comprise a first particle that comprises a first primer having a 5′ end and a 3′ end, where the first primer is linked to the first particle via the 5′ end of the first primer, and where the first primer exhibits sequence homology to a target nucleic acid strand at a 5′ end of the target nucleic acid strand. The set of particles can also comprise a second particle that comprises a second primer having a 5′ end and a 3′ end, where the second primer is linked to the second particle via the 5′ end of the second primer, and where the second primer exhibits sequence homology to a complement nucleic acid strand of the target nucleic acid strand at a 5′ end of the complement nucleic acid strand. Moreover, a sequence of the target nucleic acid strand to which the first primer exhibits homology may be separated from a sequence of the target nucleic acid strand to which the second primer exhibits complementarity by at least about 10 nucleotides.

In some embodiments, the sequence on the target nucleic acid strand to which the first primer exhibits homology may be separated from the sequence on the target nucleic acid strand to which the second primer exhibits complementarity by at least about 25 nucleotides, at least about 50 nucleotides, or at least about 100 nucleotides. In some embodiments, the first particle and/or the second particle may be contained in a partition such as, for example, a droplet in an emulsion, a well or a vessel. In cases where the partition is a well, the well may be among an array of wells.

In some embodiments, the first particle may comprise at least two of the first primer and/or the second particle may comprise at least two of the second primer. In some embodiments, the first and/or second particle may be selected from the group consisting of solid particles, porous particles, nanoparticles, beads, microparticles, metal particles, magnetic particles, semiconductor particles, polymeric particles, and nucleic acid particles. In some embodiments, the first and/or second particle may comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof. In some embodiments, the first particle and/or second particle may have a dimension of about 0.5 nm to about 100 nm. In some embodiments, the first particle and/or second particle may have a dimension of about 1 nm to about 20 nm.

An additional aspect of the disclosure provides an isolated nucleic acid complex. The isolated nucleic acid complex can comprise a double-stranded nucleic acid molecule having at least a first strand and a second strand that is at least partially complementary to the first strand. The first strand can be coupled to a first particle at a 5′ end of the first strand and the second strand can be coupled to a separate second particle at a 5′ end of the second strand.

In some embodiments, the double-stranded nucleic acid molecule can comprise a first end sequence and a second end sequence, wherein the first end sequence is complexed with the first particle via a first capture sequence linked to the first particle. The second end sequence can be complexed with the second particle via a second capture sequence linked to the second particle. Moreover, the first capture sequence can be at least partially complementary to the first end sequence and the second capture sequence can be at least partially complementary to the second end sequence.

In some embodiments, the first capture sequence may be linked to the first particle at a 3′ end of the first capture sequence and/or the second capture sequence may be linked to the second particle at a 3′ end of the second capture sequence. In some embodiments, the first strand may be covalently linked to the first particle at the 5′ end of the first strand and/or the second strand may be covalently linked to the second particle at the 5′ end of the second strand. In some embodiments, the nucleic acid complex may not be immobilized to a support.

In some embodiments, the isolated nucleic acid complex may further comprise at least three particles, at least five particles or at least ten particles each of which particles can be linked to another particle through an additional double-stranded nucleic acid molecule that is substantially the same as the double-stranded nucleic acid molecule. In some embodiments, the first particle and the second particle may comprise a dimension of about 0.5 nanometers (nm) to about 100 nanometers. In some embodiments, the first particle and the second particle may comprise a dimension of about 1 nm to about 20 nm.

In some embodiments, the nucleic acid complex may be contained in a partition such as, for example, a droplet in an emulsion, a well or a vessel. In cases where the partition is a well, the well may be a well among an array of wells. In some embodiments, the first and/or second particle may be selected from the group consisting of solid particles, porous particles, nanoparticles, beads, microparticles, metal particles, magnetic particles, semiconductor particles, polymeric particles, and nucleic acid particles. In some embodiments, the first and/or second particle may comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof.

An additional aspect of the disclosure provides a kit for assaying the presence or absence of a target nucleic acid strand in a sample having or suspected of having the target nucleic acid strand. The kit can comprise a first particle and a second particle. The first particle may comprise a first primer having a first nucleic acid sequence that exhibits sequence homology to a portion of a target nucleic acid strand and the second particle may comprise a second primer having a second nucleic acid sequence that exhibits sequence homology to a portion of a complement nucleic acid strand of the target nucleic acid strand. Moreover, the first nucleic acid sequence may be different than the second nucleic acid sequence. The kit can also comprise instructions for using the first and second particles to identify the presence or absence of the target nucleic acid strand in the sample via a primer extension reaction.

In some embodiments, the first and/or second particles may be contained in a vessel. In some embodiments, the first and/or second particle may be selected from the group consisting of solid particles, porous particles, nanoparticles, beads, microparticles, metal particles, magnetic particles, semiconductor particles, polymeric particles, and nucleic acid particles. In some embodiments, the first and/or second particle may comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof.

In some embodiments, the kit may further comprise one or more reagents suitable for generating a water-in-oil emulsion. Such reagents include, for example, a buffer, oil, and a surfactant. In some embodiments, the kit may further comprise a detectable species that permits the identification of the target nucleic acid strand. The detectable species may be, for example, an optically-responsive species. In some embodiments, the kit may further comprise reagents necessary for performing the primer extension reaction. Such reagents include, for example, a polymerase and nucleotides.

An additional aspect of the disclosure provides a method for assaying the presence of a target nucleic acid molecule in a sample having or suspected of having the target nucleic acid molecule. The method can comprise subjecting the sample to a nucleic acid amplification reaction in a reaction mixture under conditions to yield an amplified target nucleic acid molecule linked to at least a first particle and a second particle, which amplified target nucleic acid molecule comprises at least a portion of a sequence of the target nucleic acid molecule. The reaction mixture can comprise the first particle having a first primer with a 5′ end and a 3′ end. The first primer can be linked to the first particle via the 5′ end of the first primer and the first primer can exhibit sequence homology to a strand of the target nucleic acid molecule at a 5′ end of the strand of the target nucleic acid molecule. The reaction mixture can also comprise the second particle having a second primer with a 5′ end and a 3′ end. The second primer can be linked to the second particle via the 5′ end of the second primer and the second primer can exhibit sequence homology to a complement strand of the strand of the target nucleic acid molecule at a 5′ end of the complement strand. A sequence on the strand of the target nucleic acid molecule to which the first primer exhibits homology may be different than a sequence of the complement strand to which the second primer exhibits homology. Moreover, the first primer and the second primer can be adapted to be extended in a primer extension reaction to form a copy of the target nucleic acid molecule or a portion thereof.

In some embodiments, the reaction mixture may further comprise a polymerase. Such a polymerase may extend the 3′ end of the first primer in a primer extension reaction to form a copy of the strand of the target nucleic acid or a portion thereof. Moreover, such a polymerase may extend the 3′ end of the second primer in a primer extension reaction to form the complement strand or a portion thereof. In some embodiments, the reaction mixture may further comprise an optically-responsive species such as, for example, a dye or fluorescent protein. In some embodiments, the reaction mixture may be contained in a partition such as, for example, a droplet in an emulsion, a well or a vessel. In cases where the partition is a well, the well may be a well in an array of wells.

In some embodiments, the method can further comprise detecting the amplified target nucleic acid molecule linked to the first particle and second particle. The amplified target nucleic acid molecule can detected, for example, optically, electrically, physically, spectroscopically, electrochemically or electrostatically. In some embodiments, a plurality of amplified target nucleic acid molecules is detected. In some embodiments, the target nucleic acid molecule may be single-stranded.

An additional aspect of the disclosure provides a method for generating a nucleic acid complex comprising a target nucleic acid molecule. The method can comprise, in a reaction mixture, amplifying the target nucleic acid molecule with a forward primer and a reverse primer, under conditions that yield the nucleic acid complex. The nucleic acid complex can comprise an amplified target nucleic acid molecule, where the amplified target nucleic acid molecule comprises a first strand and a second strand that is at least partially complementary to the first strand. The first strand can be coupled to a first particle at a 5′ end of the first strand and the second strand can be coupled to the second particle at a 5′ end of the second strand.

In some embodiments, the forward primer may be linked to the first particle and the reverse primer may be linked to the second particle. In some embodiments, the reaction mixture may comprise the first particle and the second particle. In some embodiments, the method may further comprise amplifying the target nucleic acid molecule with a third particle linked thereto the forward primer or the reverse primer to yield the nucleic acid complex. The nucleic acid complex can further comprise the third particle coupled to the first particle and/or the second particle via an additional amplified target nucleic acid molecule.

In some embodiments, the method may further comprise detecting the nucleic acid complex. The nucleic acid complex may be detected, for example, optically, spectroscopically, physically, electrically, electrochemically or electrostatically. In some embodiments, the first particle and the second particle may be metallic particles and detection of the nucleic acid complex may be effected by visual examination. In some embodiments, the method may further comprise detecting the nucleic acid complex with the aid of an optically-responsive species.

In some embodiments, the method may further comprise isolating the nucleic acid complex by centrifugation, magnetic separation, sedimentation, filtration, chromatography, capillary action or affinity capture of the nucleic acid complex. In some embodiments, the method may further comprise isolating the nucleic acid complex by affinity capture of the nucleic acid complex on a support. In some embodiments, the reaction mixture may be contained in a partition such as, for example, a droplet in an emulsion or a well. In some embodiments, the method may further comprise releasing the nucleic acid complex from the partition and detecting the released nucleic acid complex.

Another aspect of the disclosure provides a method for identifying the presence or absence of a target nucleic acid molecule in a sample. The method can comprise providing a solution that is suspected of containing the target nucleic acid molecule. The target nucleic acid molecule can comprise a first nucleic acid strand linked to a first particle, and a second nucleic acid strand linked to a second particle. The second strand can be hybridized to the first strand via sequence complementarity to yield a nucleic acid complex. The method can further comprise detecting a signal indicative of the presence or absence of the nucleic acid complex in the solution, thereby identifying the presence of the target nucleic acid molecule.

In some embodiments, providing the solution can comprise providing the solution to a detector and the detector can detect the signal indicative of the presence or absence of the nucleic acid complex in the solution. In some embodiments, the signal can be indicative of an optical property, physical property, electrical property, electrostatic property, or electrochemical property of the nucleic acid complex. In some embodiments, the first particle and the second particle may be metallic particles and the signals can be detected by visual examination. In some embodiments, the signal may be generated from the activity of an optically-responsive species such as, for example, a dye or fluorescent protein. Examples of a dye include SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin.

In some embodiments, the solution may be contained within a partition such as, for example, a droplet in an emulsion or a well (e.g., a well in an array of wells). In some embodiments, the solution may comprise a plurality of nucleic acid complexes that comprise two or more particles. In some embodiments, the nucleic acid complex may not be affixed to a support.

Another aspect of the disclosure provides a method for assaying a sample for the presence or absence of a target nucleic acid sequence. The method can comprise receiving a request to assay the sample for the presence or absence of the target nucleic acid sequence. The method can further comprise assaying the sample for the presence or absence of the target nucleic acid sequence by detecting at least one nucleic acid complex that comprises a double-stranded nucleic acid molecule linked to at least a first particle and a second particle. The double-stranded nucleic molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to the first single-stranded nucleic acid molecule. At least one of the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule can comprise the target nucleic acid sequence. Moreover, the method can further comprise generating a report that is indicative of the presence or absence of the target nucleic acid sequence in the sample.

In some embodiments, assaying the sample for the presence or absence of the target nucleic acid sequence can comprise detecting a signal that is indicative of the presence or absence of the nucleic acid complex. The signal can be, for example, indicative of an optical property, physical property, electrical property, spectroscopic property, electrostatic property, or electrochemical property of the nucleic acid complex. In some embodiments, the signal may be generated from the activity of an optically-responsive species such as, for example, a dye. Examples of dyes include SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin.

In some embodiments, the nucleic acid complex may be detected in a partition such as, for example, a droplet in an emulsion or a well (e.g., a well in an array of wells). In some embodiments, the nucleic acid complex may comprise a plurality of double-stranded nucleic acid molecules linked to greater than two particles. In some embodiments, the report is an electronic report that can be presented on a user interface of an electronic display of an electronic device.

An additional aspect of the disclosure provides a method for generating a nucleic acid complex comprising a target nucleic acid molecule. The method can comprise, in a reaction mixture, amplifying the target nucleic acid molecule with a forward primer and a reverse primer to yield an amplified target nucleic acid molecule. The forward primer and the reverse primer can each comprise a 3′ end capable of being extended in a primer extension reaction and a hairpin structure. The amplified target nucleic acid molecule can comprise a first strand comprising a first overhang sequence at one end of the amplified target nucleic acid molecule and a second strand comprising a second overhang sequence at the other end of the amplified target nucleic acid molecule. Moreover, the method can further comprise contacting the amplified target nucleic acid molecule with a first particle and a second particle to yield a nucleic acid complex. The nucleic acid complex can comprise the amplified target nucleic acid molecule that is complexed with the first particle via sequence complementarity between the first overhang sequence and a first capture sequence linked to the first particle; and the second particle via sequence complementarity between the second overhang sequence and a second capture sequence linked to the second particle.

In some embodiments, the forward primer and the reverse primer further may comprise a spacer region which cannot be copied via a primer extension reaction. Such a spacer region may be selected from the group consisting of C3 spacer, an abasic site, a carbonaceous linker, polyethylene glycol (PEG) and combinations thereof. In some embodiments, the method may further comprise ligating the first strand to the second capture sequence and/or ligating the second strand to the first capture sequence.

Another aspect of the disclosure provides a method for nucleic acid amplification. The method can comprise annealing a forward primer linked to a first particle to a nucleic acid strand and a reverse primer linked to a second particle to a complement strand of the nucleic acid strand. The method can further comprise extending the forward primer and the reverse primer in a template-directed manner to yield a first double-stranded nucleic acid molecule linked to the first particle and a second double-stranded nucleic acid molecule linked to the second particle. The method may further comprise denaturing the first double-stranded nucleic acid molecule and the second double-stranded nucleic acid molecule to generate a first single-stranded molecule linked to the first particle and a second single-stranded molecule linked to the second particle. The method can further comprise repeating annealing, extending and denaturing as described above can be repeated by annealing the forward primer to the second single-stranded molecule and annealing the reverse primer to the first single-stranded molecule to yield a nucleic acid complex comprising an amplified double-stranded nucleic acid molecule. The amplified double-stranded nucleic acid molecule can linked at one end to the first particle and linked at its other end to the second particle.

In some embodiments, the method is performed in a partition. In some embodiments, the method may further comprise amplifying the nucleic acid strand with a third particle linked thereto the forward primer or the reverse primer to yield the nucleic acid complex that further comprises the third particle coupled to the first particle and/or the second particle via an additional amplified double-stranded nucleic acid molecule. In some embodiments, the method can further comprise detecting the nucleic acid complex optically, spectroscopically physically, electrically, electrochemically, or electrostatically.

An additional aspect of the disclosure provides a system for analyzing the content(s) of a solution. The system can comprise a detection cell that is adapted to contain or direct a solution containing a nucleic acid complex that comprises a double-stranded nucleic acid molecule that is linked to at least a first particle and a second particle. The double-stranded nucleic molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule. The system can further comprise a detector that can be linked to the detection cell and detects signals indicative of the presence or absence of a nucleic acid complex in the solution. Moreover, the system can further comprise a computer processor that can be linked to the detector and programmed to receive signals from the detector, which signals are indicative of the presence or absence of the nucleic acid complex. The computer processor can also be programmed to determine if the nucleic acid complex is present or absent in the solution based on the detected signals.

In some embodiments, the detection cell may comprise a vessel or an array of wells. In some embodiments, the detection cell may comprise a support. In some embodiments, the detection cell may comprise a fluid flow path that may be, for example, a microfluidic channel. In some embodiments, the detector may be selected from the group consisting of an optical detector, a spectroscopic detector and an electrochemical detector.

In some embodiments, the detection cell may further comprise the solution. In some embodiments, the solution may be contained within a partition such as, for example, a well (e.g., a well in an array of wells) or a droplet in an emulsion. In some embodiments, the nucleic acid complex may comprise a plurality of double-stranded nucleic acid molecules linked to greater than two particles.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “Fig.” herein), of which:

FIG. 1 is a schematic depiction of an example method of isolating and detecting a nucleic acid complex and/or a nucleic acid molecule of a nucleic acid complex;

FIG. 2 is a schematic depiction of an example method of isolating and detecting a nucleic acid complex and/or nucleic acid molecule of a nucleic acid complex;

FIG. 3 (panel A) and FIG. 3 (panel B) are schematic depictions of an example primer (SEQ ID NO: 1) used in nucleic acid amplification;

FIG. 3 (panel C) is a schematic depiction of an example method of using an example primer in nucleic acid amplification;

FIG. 4 is a schematic depiction of an example method for nucleic acid amplification to yield a nucleic acid complex;

FIG. 5 is a schematic depiction of an example method for nucleic acid amplification to yield a nucleic acid complex;

FIG. 6 is a schematic depiction of an example method for nucleic acid amplification to yield a nucleic acid complex and isolation of the nucleic acid complex;

FIG. 7 is a schematic depiction of an example method for multiplex nucleic acid amplification;

FIG. 8 is a schematic depiction of an example method for multiplex nucleic acid amplification;

FIG. 9 is a schematic depiction of an example method for processing a nucleic acid complex for completing a sequencing reaction; and

FIG. 10 is a schematic depiction of an example computer system that comprises a computer processor.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a nucleic acid molecule” includes a plurality of nucleic acid molecules, including mixtures thereof.

As used herein, the terms “amplifying” and “amplification” are used interchangeably and generally refer to producing one or more copies of a nucleic acid.

As used herein, the terms “anneal” and “annealing” generally refer to the binding of one nucleic acid molecule (e.g., a primer) with another nucleic acid molecule (e.g., a template nucleic acid molecule) via complementarity between the nucleic acid molecules.

As used herein, the term “capture sequence” generally refers to a nucleic acid sequence that is associated with a support and is capable of coupling (e.g., hybridizing) with a nucleic acid molecule via sequence complementarity such that the coupling of the nucleic acid molecule with the capture sequence immobilizes the nucleic acid molecule to the support.

“Complementarity” or “complementary” generally refer to the ability of a nucleic acid molecule to form hydrogen bond(s) with another nucleic acid molecule by either Watson-Crick or other types of base-pairing. “Partially complementary” generally means that a portion of a first nucleic acid sequence will hydrogen bond with a portion of a second nucleic acid sequence.

As used herein, the terms “denaturing” and “denaturation” are used interchangeably and generally refer to the full or partial unwinding of the helical structure of a double-stranded nucleic acid, and in some embodiments the unwinding of the secondary structure of a single stranded nucleic acid.

As used herein, a “detectable species” generally refers to a composition that yields a detectable signal, the presence or absence of which can be used to detect the presence of a nucleic acid and/or copies of a nucleic acid. In some embodiments, a detectable species may be an optically-responsive species. As used herein, the term “optically-responsive species” generally refers to a detectable species that yields a detectable signal in the presence (or absence) of electromagnetic radiation, such as, for example, light. Examples of detectable moieties are provided elsewhere herein.

As used herein, the term “linked” and “coupled” are used interchangeably and generally refer to the association of two species. Two species may be linked or coupled in any suitable way including direct linkage or coupling (e.g., direct attachment between species), indirect linkage or coupling (e.g., attachment via a linker coupled to both species), covalent attachment, or non-covalent attachment (e.g., hybridization between nucleic acid molecules, binding of members of an affinity binding pair, ionic interactions, hydrophobic interactions, Van der Waals forces, etc.) and combinations thereof.

As used herein, the term “melting temperature” (T_(m)) generally refers to the temperature at which two single-stranded nucleic acid molecules that are hybridized and form a double-stranded molecule dissociate from each other. In some embodiments, a melting temperature can refer to a temperature at which about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical nucleic acid strands of a population of identical double-stranded nucleic acid molecules dissociate from their complement strands. For example, the melting temperature of a primer may refer to the temperature at which about half of the molecules of the primer in a population of identical primers hybridized to a nucleic acid molecule dissociate from their complementary sequence on their respective nucleic acid molecules. In some embodiments, the melting temperature of a primer may be from about 20° C. to about 80° C. In some embodiments, the melting temperature of a primer may be from about 25° C. to about 75° C. In some embodiments, the melting temperature of a primer may be from about 25° C. to about 60° C. In some embodiments, the melting temperature of a primer may be about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., or higher.

As used herein, the terms “nucleic acid” and “nucleic acid molecule” are used interchangeably and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs), or analogs thereof. Nucleic acids may have any three dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a peptide nucleic acid (PNA), coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs such as, for example, locked nucleic acids (LNA), fluorinated nucleic acids (FNA), bridged nucleic acids and thio-nucleotides. If present, modifications to the nucleotide structure may be made before or after assembly of the nucleic acid. The sequence of nucleotides of a nucleic acid may be interrupted by non-nucleotide components, such as, for example a linker or other type of spacer. A nucleic acid may be further modified after polymerization, such as by conjugation or binding with a detectable species. In some embodiments, a nucleic acid may be a primer that, in some embodiments, can be used to amplify another nucleic acid molecule.

As used herein, the term “nucleic acid complex” generally refers to a complex comprising a plurality of particles linked together via one or more double-stranded nucleic acid molecules. A nucleic acid complex may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more particles associated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more double-stranded nucleic acid molecules.

As used herein, the term “primer” generally refers to a nucleic acid molecule that is capable of hybridizing with a template nucleic acid molecule and capable of being extended in a template-directed manner via the template nucleic acid molecule.

A “primer extension reaction” generally refers to the binding (e.g., “annealing”) of a primer to a strand of nucleic acid, followed by incorporation of nucleotides to the primer (e.g., “extension” of or “extending” the primer), using the strand of nucleic acid as a template. A primer extension reaction may be completed with the aid of an enzyme, such as, for example a polymerase.

As used herein, the term “reaction mixture” generally refers to a composition comprising one or more reagents necessary to complete denaturation of a double-stranded nucleic acid molecule, annealing of a primer to a strand of nucleic acid, extension of the primer in a primer extension reaction and/or nucleic acid amplification, with non-limiting examples of such reagents that include one or more primers having specificity for a target nucleic acid, a polymerase, suitable buffers, co-factors (e.g., divalent and monovalent cations), nucleotides (e.g., deoxyribonucleotides (dNTPs)), any other enzymes, surfactants and additives that can modulate nucleic acid hybridization. In some embodiments, a reaction mixture can also comprise one or more detectable species.

As used herein, the term “sequence homology” generally refers to a degree of sequence congruence between two or more nucleic acid molecules. For example, a nucleic acid molecule can exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with one or more other nucleic acid molecules when optimally aligned with the one or more nucleic acid molecules. Sequence homology may occur at the 5′ ends of a plurality of nucleic acid molecules (e.g., two nucleic acid molecules may have a common nucleic acid sequence each at the 5′ end of each nucleic acid molecule), at the 3′ ends of a plurality of nucleic acid molecules (e.g., two nucleic acid molecules may have a common nucleic acid sequence at the 3′ end of each nucleic acid molecule); or may occur at different locations of different nucleic acid molecules (e.g., one nucleic acid molecule may have a common nucleic acid sequence at its 5′ end and another nucleic acid molecule may have the common nucleic acid sequence between its 5′ end and 3′ end).

As used herein, the term “support” generally refers to a species on which another species can be immobilized. Non-limiting examples of supports include a particle, a surface of a well, a surface of a vessel, a solid surface, a planar surface, a surface of an array, a porous surface (e.g., a micro-cavity of a porous surface), a resin (e.g., a resin in a column) and a fiber (e.g., a fiber in a membrane or support). Moreover, a support can comprise any suitable material with non-limiting examples that include a metal, a metal oxide, carbonaceous materials and polymeric species. A support may be used, for example, to immobilize a nucleic acid molecule and/or may be used to immobilize a nucleic acid complex.

As used herein, the terms “target nucleic acid” and “target nucleic acid molecule” are used interchangeably and generally refer to a nucleic acid molecule in a starting population of nucleic acid molecules having a target sequence whose presence, amount, and/or nucleotide sequence, or changes in one or more of these, are desired to be determined. In some embodiments, a target nucleic acid molecule may be double-stranded. In some embodiments, a target nucleic acid molecule may be single-stranded. In general, the term “target nucleic acid strand” refers to a single-stranded target nucleic acid molecule. In general, the term “target nucleic acid sequence” refers to a nucleic acid sequence on a strand of target nucleic acid. A target nucleic acid molecule or target nucleic acid sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others. The target nucleic acid sequence or target nucleic acid molecule may be a target nucleic acid sequence or target nucleic acid molecule from a sample or a secondary target such as a product of an amplification reaction.

Various aspects of the disclosure provide sets of particles for nucleic acid amplification and isolated nucleic acid complexes that comprise nucleic acid molecules linked to a plurality of particles. Sets of particles can include a first and second particle each associated with a nucleic acid molecule such as primer and/or capture sequence. Isolated nucleic acid complexes can include a first particle and a second particle, which first and second particles are linked via a double-stranded nucleic acid molecule linked to both the first particle and the second particle.

In one aspect, the disclosure provides a set of particles for nucleic acid amplification that includes a first particle and a second particle. The first particle may comprise a first primer, having a 5′ end and a 3′ end and linked to the first particle via the 5′ end of the first primer. The first primer can exhibit sequence homology to a target nucleic acid strand at a 5′ end of the target nucleic acid strand. The second particle may comprise a second primer, having a 5′ end and a 3′ end and linked to the second particle via the 5′ end of the second primer. The second primer can exhibit sequence homology to a complement nucleic acid strand of the target nucleic acid strand at a 5′ end of the complement nucleic acid strand. In addition, a sequence on the target nucleic acid strand to which the first primer exhibits homology can be different than a sequence on the complement nucleic acid strand to which the second primer exhibits homology.

In another aspect, the disclosure provides a set of particles for nucleic acid amplification that includes a first particle and a second particle. The first particle may comprise a first primer, having a 5′ end and a 3′ end and linked to the first particle via the 5′ end of the first primer. The first primer can exhibit sequence homology to a target nucleic acid strand at a 5′ end of the target nucleic acid strand. The second particle may comprise a second primer, having a 5′ end and a 3′ end and linked to the second particle via the 5′ end of the second primer. The second primer can exhibit sequence homology to a complement nucleic acid strand of the target nucleic acid strand at a 5′ end of the complement nucleic acid strand. In addition, a sequence of the target nucleic acid strand to which the first primer exhibits homology may be separated from a sequence of the target nucleic acid strand to which the second primer exhibits complementarity by at least about 10 nucleotides.

In any set of particles described herein, a sequence on the target nucleic acid strand to which the first primer exhibits homology may be separated from a sequence on the target nucleic acid strand to which the second primer exhibits complementarity by at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides. Additionally, in any set of particles described herein, the first primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the target nucleic acid strand at a 5′ end of the target nucleic acid strand when optimally aligned with the target nucleic acid strand. Similarly, in any set of particles described herein, the second primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the complement nucleic acid strand of the target nucleic acid strand at a 5′ end of the complement nucleic acid strand when optimally aligned with the complement nucleic acid strand.

In any set of particles described herein, the first particle may comprise at least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more molecules of the first primer. Moreover, the second particle may comprise at least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more molecules of the second primer. In any set of particles described herein, the first particle and second particle may also comprise the second primer and first primer, respectively. Moreover, the length of the first primer and the second primer linked to the first and second particles, respectively, may vary depending upon the particular application. For example, the length of the first primer or second primer may be about 1 nucleotide to about 100 nucleotides in length; about 5 to about 50 nucleotides in length; about 5 to about 30 nucleotides in length; or about 15 to about 30 nucleotides in length. In some embodiments, the length of the first primer or the second primer may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides in length.

Additionally, the first primer may be linked to the first particle and the second primer may be linked to the second particle, including via direct attachment or indirect attachment via, for example, at least one linker. Non-limiting examples of such linkers include a polymeric species, a nucleic acid, a phosphate moiety, an amino acid, a peptide, a hydrocarbon chain, a polysaccharide, a polyethylene glycol (PEG) and combinations thereof. Direct and indirect attachments between primers and particles may be via covalent bonds (e.g., covalent bonds between primer and particle, covalent bonds between linker and particle and/or primer), or non-covalent interactions (e.g., hybridization between nucleic acid molecules, binding of members of an affinity binding pair (e.g., streptavidin/biotin), ionic interactions, hydrophobic interactions, Van der Waals forces, etc.) and combinations thereof.

Moreover, the particles in any set of particles described herein may be linked to respective primers via any suitable chemistry or combination of chemistries (e.g., for functionalizing particles with groups that may be used for linking primers and particles). Non-limiting examples of such chemistries include thiol chemistry, phosphonic acid chemistry (e.g., in cases where a particle comprises a metal oxide), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry, aldehyde chemistry, epoxy chemistry, homobifunctional cross-linker chemistry (e.g., via amine functional groups, via thiol functional groups), heterobifunctional cross-linker chemistry (e.g., via amine functional groups, via thiol functional groups), 1,4-phenylenedisiothiocyanate (PDC) chemistry, organosilanization, ionized gas treatments, UV irradiation, click chemistry, diacetone acrylamide crosslinking and combinations thereof.

In any set of particles described herein, the 3′ end of the first primer may be adapted to be extended in a primer extension reaction to form a copy of the target nucleic acid strand or a portion thereof. Moreover, the 3′ end of the second primer may be adapted to be extended in a primer extension reaction to form the complement nucleic acid strand or a portion thereof. In addition, any set of particles described herein may also comprise more particles than a first particle and a second particle, where each additional particle comprises the first and/or second primer. For example, a set of particles described herein may comprise about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000, 500000000, 1000000000 or more particles each comprising the first and/or second primer.

In another aspect, the disclosure provides an isolated nucleic acid complex. The isolated nucleic acid complex may comprise a double-stranded nucleic acid molecule having at least a first strand and a second strand that is at least partially complementary to the first strand. The first strand can be coupled to a first particle at a 5′ end of the first strand and the second strand can be coupled to a separate second particle at a 5′ end of the second strand.

In some embodiments, the first strand of the double-stranded nucleic acid molecule may be coupled to the first particle and the second strand of the double-stranded nucleic acid molecule may be coupled to the second particle, including via direct attachment or indirect attachment as described elsewhere herein for linking of primers and particles. Direct and indirect attachments between the first and second strands and respective particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein. For example, the first strand may be covalently linked to the first particle at a 5′ end of the first strand and/or the second strand may be covalently linked to the second particle at a 5′ end of the second strand. Furthermore, in some embodiments, the isolated nucleic acid complex may not be immobilized to a support and instead, for example, suspended or free-floating in a liquid medium.

In some embodiments, the double-stranded nucleic acid molecule may include a first end sequence and a second end sequence. Each of the first and second end sequences can be a single-stranded nucleic acid sequence linked to the 5′ or 3′ ends of the first and second strands, respectively, of the double-stranded nucleic acid molecule. The first end sequence can be complexed with the first particle via a first capture sequence linked to the first particle and the second end sequence can be complexed with the second particle via a second capture sequence linked to the second particle. The first capture sequence can be an oligonucleotide linked to the first particle and configured to hybridize with the first end sequence of the double-stranded nucleic acid molecule via sequence complementarity. The first capture sequence may be fully or at least partially complementary to the first end sequence. Additionally, the second capture sequence can be an oligonucleotide linked to the second particle and configured to hybridize with the second end sequence of the double-stranded nucleic acid molecule via sequence complementarity. The second capture sequence may be fully or at least partially complementary to the second end sequence. In some embodiments, the first and/or second capture sequences may comprise a locked nucleic acid (LNA).

In some embodiments, the first capture sequence may be linked to the first particle at a 3′ end of the first capture sequence and/or the second capture sequence may be linked to the second particle at a 3′ end of the second capture sequence. Additionally, the first capture sequence may be linked to the first particle and the second capture sequence may be linked to the second particle, including via direct attachment or indirect attachment as described elsewhere herein for linking of primers and particles. Direct and indirect attachments between capture sequences and particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein.

In some embodiments, the first particle may comprise at least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more molecules of the first capture sequence. Moreover, the second particle may comprise at least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more molecules of the second capture sequence. In some embodiments, the first particle and second particle may also comprise the second capture sequence and first capture sequence, respectively.

Moreover, the length of the first and second capture sequences may vary depending upon the particular application. For example, the length of the first or second capture sequence may be about 1 nucleotide to about 100 nucleotides in length; about 5 to about 50 nucleotides in length; about 5 to about 30 nucleotides in length; or about 15 to about 30 nucleotides in length. In some embodiments, the length of the first or second capture sequence may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides in length.

The length of the first and second end sequences may vary depending upon the particular application. For example, the length of the first or second end sequence may be about 1 nucleotide to about 100 nucleotides in length; about 5 to about 50 nucleotides in length; about 5 to about 30 nucleotides in length; or about 15 to about 30 nucleotides in length. In some embodiments, the length of the first or second end sequence may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides in length.

In some embodiments, the isolated nucleic acid complex may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more particles associated with about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more double-stranded nucleic acid molecules. Each particle of the isolated nucleic acid complex can be linked to another particle in the isolated nucleic acid complex through an additional double-stranded nucleic acid molecule that is substantially the same as the double-stranded nucleic acid. The term “substantially the same” as used herein refers to a degree of sequence homology between the double-stranded nucleic acid molecule and the additional double-stranded nucleic acid molecule of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

Any particle, set of particles, or nucleic acid complex described herein may comprise any suitable type of particle. Non-limiting examples of particle types include solid particles, porous particles, nanoparticles (e.g., nanotubes, nanorods, nanoshells, spherical nanoparticles), microparticles, beads, metal particles, magnetic particles (e.g., magnetic nanoparticles), semiconductor particles (e.g., quantum dots), polymeric particles, nucleic acid particles (e.g., DNA-containing particles, RNA-containing particles, etc), fluorescent particles, colorimetric particles, composites thereof and combinations thereof. Moreover, any particle, particle of a set of particles, or particle of an isolated nucleic acid complex described herein may comprise any suitable material or plurality of materials (e.g., particles of one material coated with another material, particles made of composite materials, etc.). Non-limiting examples of materials that a particle can comprise include metals (e.g., silver, gold, platinum, copper, palladium), metal oxides (e.g., Al₂O₃, NiO, Fe₂O₃, ZrO₂, MoO₃, CeO₂, Y₂O₃, TiO₂, ZnO, SnO, ITO, Co₃O₄), polymers (e.g., polyacrylamide, polystyrene, dextrose, polyaniline, polypyrrole, polyacetylene, a fluorescent protein, carbon, composites thereof and combinations thereof. In some embodiments, a first particle and a second particle in a set of particles and/or a nucleic acid complex may comprise the same materials. In some embodiments, a first particle and a second particle in a set of particles and/or a nucleic acid complex may comprise different materials. For example, a set of particles or nucleic acid complex may comprise a first particle that comprises gold and a second particle that comprises silver.

Any particle, particle of a set of particles, or particle of an isolated nucleic acid complex described herein may have varied particle size depending upon, for example, the particular application envisioned. In general, “particle size” as used herein generally refers to the size of a dimension of a particle. Such a dimension, for example, may be a diameter, a circumference, a perimeter, a hydrodynamic diameter, a radius, a length, a width, or a depth of a particle. In some embodiments, a particle may have a dimension of about 0.1 nanometers (nm) to about 100 nm; about 0.5 nm to about 100 nm; about 1 nm to about 20 nm; about 1 nm to about 10 nm; or about 1 nm to 5 nm. In some embodiments a particle may have a dimension of at most 100 nm, at most 50 nm, at most 20 nm, at most 10 nm or at most 5 nm. In some embodiments, a particle may have a dimension of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 nm or the dimension may be larger or smaller depending on the particular particle.

Any particle, particle of a set of particles, or particle of an isolated nucleic acid complex described herein may have any suitable shape/configuration that may be a regular shape/configuration or may be an irregular shape/configuration. Non-limiting examples of particle shapes include spheres, rods, hollow shells, tubes, elliptical particles, core-shell particles (e.g., coated particles), agglomerations of smaller particles and combinations thereof. Moreover, any particle, particle of a set of particles, or particle of an isolated nucleic acid complex described herein may or may not be linked with a support. In embodiments where a particle, particle of a set of particles or particle of an isolated nucleic acid complex is not linked with a support, the particle(s) may be, for example, free-floating or suspended in a liquid medium (e.g., aqueous medium).

In some embodiments, a particle, particle of a set of particles, or particle of an isolated nucleic acid complex described herein may comprise an affinity capture agent that is configured to bind to a binding partner. A suitable affinity of the affinity capture agents for its binding partner generally can allow the two species to bind. Affinity capture agents may be useful in isolating a particle, particle of a set of particles or a nucleic acid complex as described elsewhere herein. Non-limiting examples of affinity capture agents include a non-nucleic acid member of a binding pair (e.g., streptavidin or biotin from a streptavidin-biotin binding pair), a capture sequence (e.g., a capture sequence comprising a locked nucleic acid (LNA)), antibody-antigen pairs (e.g., anti-fluorescein antibody and fluorescein, anti digoxigenin antibody and digoxigenin), leptin-sugar pairs, aptamers and binding pairs, polypeptides with their binding pairs and or other types of polymers with their binding pairs.

For example, a particle, a particle of a set of particles, or a particle of an isolated nucleic acid complex described herein may comprise a free primer or a capture sequence that has not coupled with another nucleic acid molecule. In another example, a particle, a particle of a set of particles, or a particle of an isolated nucleic acid complex described herein may comprise a member of a binding pair such as, for example, one or both of streptavidin or biotin. Additionally, an affinity capture agent may be linked to a particle, including via direct attachment or indirect attachment as described elsewhere herein for linking of primers and particles. Direct and indirect attachments between affinity capture sequences and particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein.

Furthermore, any particle, set of particles, or isolated nucleic acid complex described herein may be contained in a partition. The partition may be any suitable type of partition with non-limiting examples of partitions that include a droplet (e.g., a droplet in an emulsion, such as, for example, a water-in-oil emulsion or oil-in-water emulsion), a vessel (e.g., any suitable type of tube, a capillary tube, a centrifuge tube, a cuvette, a pipette tip, a bag, a box, a container) and combinations thereof.

In some embodiments, a partition may be a well (e.g., a well among an array of wells such as a well in a microwell plate, a microwell, a nanowell). A well can be microfabricated in (e.g., etched into a silicon substrate) or on (e.g., depositing well materials onto a substrate) a substrate such as, for example a silicon substrate. Moreover, the surface(s) within a well can have different properties than a bulk surface substrate in or on which the well is included. For example, the well surface may be hydrophilic and the bulk substrate surface may be hydrophobic or vice versa. Additionally, in some embodiments, the surface of a well and a bulk substrate can be differentially modified with various functional groups such that they each selectively capture particular chemical agents (e.g., chemical agents having affinity for particles, a set of particles, nucleic acid complexes, etc.).

A substrate comprising wells can be placed in or be included as part of a flow-cell chamber that has an inlet and an outlet for liquid transferred. In some embodiments, such a flow-cell may also include a flat or curved inner surface where wells are patterned. A liquid phase solution can be passed through the flow-cell such that the wells are exposed to a solution. Depending on the physical and/or chemical properties of the solution and surface properties of the wells and/or other flow-cell surfaces, the solution can be partitioned into the wells or selectively prohibited from entering the wells. For example, an oil solution flowed over wells can avoid partitioning when well surfaces contain a hydrophilic surface, whereas an aqueous solution flowed over the wells can be partitioned into the wells. A dimension of a well can be any suitable dimension. For example, a dimension of a well can be about 1 nanometer (“nm”) to about 1 millimeter (“mm”), 10 nm to 1 mm, 100 nm to 1 mm, 1 micrometer (“μm”) to 1 mm, 10 μm to 1 mm or 100 μm to 1 mm.

In cases where a partition is a droplet in an emulsion, the droplet may be generated in any suitable way including bulk emulsification methods and/or with the aid of a microfluidic device. Bulk emulsification and/or microfluidic devices may also be useful in partitioning particles into droplets. Moreover, the volume of a droplet can be any suitable volume. For example, the volume of a droplet can be about 1 femtoliter (“fL”) to 100 μL microliter (“μL”), 10 fL to 10 μL, 100 fL to 10 μL, 1 picoliter (“pL”) to 1 μL, 1 nanoliter (“nL”) to 10 μL, 10 nL to 10 μL, 100 nL to 10 μL or 1 μL to 10 μL.

Additional aspects of the disclosure provide methods for assaying or identifying the presence of a target nucleic acid molecule/target nucleic acid sequence in a sample, methods for generating a nucleic acid complex comprising a target nucleic acid molecule and methods for nucleic acid amplification.

In another aspect, the disclosure provides a method for assaying the presence of a target nucleic acid molecule in a sample having or suspected of having the target nucleic acid molecule. The method comprises subjecting the sample to a nucleic acid amplification reaction in a reaction mixture under conditions to yield an amplified target nucleic acid molecule linked to at least a first particle and a second particle, where the amplified target nucleic acid molecule comprises at least a portion of a sequence of the target nucleic acid molecule. The reaction mixture may comprise the first particle and the second particle. The first particle can have a first primer, with a 5′ end and a 3′ end, linked to the first particle via the 5′ end of the first primer. The first primer can exhibit sequence homology to a strand of the target nucleic acid molecule at a 5′ end of the strand of the target nucleic acid molecule. The second particle can have a second primer, with a 5′ end and a 3′ end, linked to the second particle via the 5′ end of the second primer. The second primer can exhibit sequence homology to a complement strand of the strand of the target nucleic acid molecule at a 5′ end of the complement strand. In addition, a sequence on the strand of the target nucleic acid molecule to which the first primer exhibits homology may be different than a sequence of the complement strand to which the second primer exhibits homology. Moreover, the first primer and the second primer can be adapted to be extended in a primer extension reaction to form a copy of the target nucleic acid molecule or a portion thereof.

In some embodiments, the first primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the 5′ end of the strand of the target nucleic acid molecule when optimally aligned with the strand. Similarly, the second primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the 5′ end of the complement strand when optimally aligned with the complement strand. Furthermore, in some embodiments, the target nucleic acid molecule may be single-stranded or, in some embodiments, the target nucleic acid molecule may be double-stranded. Additionally, the first primer may be linked to the first particle and the second primer may be linked to the second particle, including via direct attachment or indirect attachment as described elsewhere herein. Direct and indirect attachments between primers and particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein.

The reaction mixture may also comprise reagents necessary for the amplification of the target nucleic acid molecule, including a polymerase and other such reagents necessary for amplification of a nucleic acid molecule described elsewhere herein. The polymerase may extend the 3′ end of the first primer in a primer extension reaction to form a copy of the strand of the target nucleic acid or a portion thereof and/or may extend the 3′ end of the second primer in a second primer extension reaction to form the complement strand or a portion thereof. Moreover, in some embodiments, the reaction mixture may be contained in a partition. Any suitable partition may be used to contain the reaction mixture including example types of partitions described elsewhere herein.

In some embodiments, the method may further comprise detecting the amplified target nucleic acid molecule linked to the first particle and the second particle. The amplified target nucleic acid molecule may be detected, including via modes of detection described elsewhere herein, such as, for example, optical detection. For example, the reaction mixture may further comprise an optically-responsive species and detection may be achieved via the optically-responsive species (e.g., a dye or other optically-responsive species described elsewhere herein). The optically-responsive species may interact with the amplified target nucleic acid molecule, such that upon successful amplification of the target nucleic acid, a detectable signal (or lack thereof) can be detected from the optically-responsive species. In some embodiments, the amplified target nucleic acid molecule may be detected by detecting either or both of the first or second particles or the assembly of the amplified target nucleic acid molecule and first and second particles (e.g., detection of a nucleic acid complex as described elsewhere herein). In some embodiments, the assembly of the amplified target nucleic acid molecule and first and second particles may be isolated prior to detection, using, for example, a mode of isolation described elsewhere herein.

In some embodiments, the method may comprise producing a plurality of amplified target nucleic acid molecules and, in some embodiments, detecting the plurality of amplified target nucleic acid molecules. In such cases, the reaction mixture may comprise more than two particles to which the plurality of amplified nucleic acid molecules can be linked. Each additional particle may comprise either or both of the first primer and the second primer.

In another aspect, the disclosure provides a method for identifying the presence or absence of a target nucleic acid molecule in a sample. The method comprises providing a solution that is suspected of containing the target nucleic acid molecule, where the target nucleic acid molecule comprises a first nucleic acid strand linked to a first particle and a second nucleic acid strand linked to a second particle. The second nucleic acid strand can be hybridized to the first nucleic acid strand via sequence complementarity to yield a nucleic acid complex. In addition, the method also comprises detecting a signal indicative of the presence or absence of the nucleic acid complex in the solution, thereby identifying the presence of the target nucleic acid molecule.

In some embodiments, the solution may be provided to a detector and the detector may detect the signal indicative of the presence or absence of the nucleic acid complex in the solution. Detection of the signal indicative of the presence or absence of the nucleic acid complex in the solution may be completed via any suitable detection method and, in some embodiments, detector. Example modes of detection and detection are described elsewhere herein. In some embodiments, the signal indicative of the presence or absence of the nucleic acid complex can be indicative of an optical property (e.g., an optically-responsive species associated with the nucleic acid complex, such as, for example, an example dye described elsewhere herein), physical property (e.g., density, size of the nucleic acid complex), an electrical property (e.g., conductance, impedance), an electrostatic property, and/or an electrochemical property of the nucleic acid complex. In some embodiments, the first and second particles may be metallic particles and detection of the signals may be detected by visual examination as described elsewhere herein.

The solution comprising the nucleic acid complex may be any suitable solution, such as, for example, a nucleic acid amplification reaction mixture as described elsewhere herein or a reaction mixture that has been processed (e.g., a reaction mixture diluted with a diluent such as water, buffer other liquid medium, or combination thereof). In some embodiments, the solution may be an aqueous solution comprising the nucleic acid complex. In some embodiments, the solution may comprise a nucleic acid complex that has been isolated from a reaction mixture, using any suitable mode of isolation including examples of isolating a nucleic acid complex described elsewhere herein. In some embodiments, the solution may be contained within a partition. Any suitable type of partition may be used to contain the solution including example types of partitions described elsewhere herein. In some embodiments, the solution may comprise nucleic acid complexes that comprise two or more particles (e.g., nucleic acid complexes that comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more particles). In some embodiments, the solution may comprise a plurality of nucleic acid complexes that comprise two or more particles, where, in some embodiments, the detected signal is indicative of the presence or absence of a plurality of nucleic acid complexes. In some embodiments, the nucleic acid complex may or may not be immobilized or affixed (e.g., linked) to a support in the solution. In embodiments where the nucleic acid complex is not immobilized or affixed to a support in the solution, the nucleic acid complex may be suspended and/or free-floating within the solution.

Additionally, the first nucleic acid strand may be linked to the first particle and the second nucleic acid strand may be linked to the second particle, including via direct attachment or indirect attachment as described elsewhere herein. Direct and indirect attachments between target nucleic acid strands and particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein.

In another aspect, the disclosure provides a method for assaying a sample for the presence or absence of a target nucleic acid sequence. The method comprises receiving a request to assay the sample for the presence or absence of the target nucleic acid sequence and assaying the sample for the presence or absence of the target nucleic acid sequence. Assaying can be completed by detecting at least one nucleic acid complex that comprises a double-stranded nucleic acid molecule linked to at least a first particle and a second particle. The double-stranded nucleic molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to the first single-stranded nucleic acid molecule. Moreover, at least one of the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule may comprise the target nucleic acid sequence. In addition, the method can further comprise generating a report that is indicative of the presence or absence of the target nucleic acid sequence in the sample.

In some embodiments, assaying may further comprise detecting a signal that is indicative of the presence or absence of the nucleic acid complex. Detection of the signal indicative of the presence or absence of the nucleic acid complex in the solution may be completed via any suitable detection method such as, for example, the example modes of detection described elsewhere herein. The detected signal may be indicative of an optical property (e.g., a signal generated from the activity of an optically-responsive species (such as, for example, a dye or other optically-responsive species described elsewhere herein) associated with the nucleic acid complex), a physical property (e.g., density or size), an electrical property (e.g., conductance, impedance), an electrostatic property, and/or an electrochemical property of the nucleic acid complex. In some embodiments, the nucleic acid complex may be detected in a partition. The nucleic acid complex may be detected in any suitable type of partition including example types of partitions described elsewhere herein. In some embodiments, the nucleic acid may be isolated prior to detection (e.g., isolated from a reaction mixture), as described elsewhere herein. In some embodiments, the nucleic acid complex may comprise a plurality of double-stranded nucleic acid molecules linked to greater than two particles as described elsewhere herein.

Moreover, the report that is generated may be any suitable type of report. A report may include any number of desired elements, with non-limiting examples that include information regarding the presence or absence of the target nucleic acid sequence in the sample, the presence or absence of the nucleic acid complex in the sample, the target nucleic acid sequence, the number of nucleic acid molecules detected in the sample, the number of copies of the target nucleic acid sequence detected in the sample, etc. The report may be provided as a printed report (e.g., a hard copy) and/or may be provided as an electronic report. An electronic report may be presented on a user interface (UI) such as a UI on an electronic display of an electronic device. In some examples, an electronic display may include a resistive or capacitive touch screen. Non-limiting examples of electronic displays include a monitor or television, a screen operatively linked with a detector, a tablet computer screen, a mobile device screen, a portable computer screen, and the like. In some embodiments a UI may be a graphical user interface (GUI) that is configured to provide a report to a user. A GUI can include textual, graphical and/or audio components. Both a printed and an electronic report may be stored in files or in databases, respectively, such that they are accessible for future use. Moreover, a report may be transmitted to a local or remote location using any suitable communication medium including, for example, a network connection, a wireless connection, and/or an internet connection.

In some embodiments, the request to assay the sample may be received from any type of requestor, with non-limiting types of requestors that include a research professional (e.g., scientist, lab technician, professor, etc.), a health-care professional (e.g., a nurse, a doctor, a physician's assistant, a medical technician, etc.), a research organization (e.g., a laboratory, a research hospital, a research center, a research institute, and academic institution, a university), a health-care organization (e.g., a hospital, a nursing home, a hospice, a medical center, a clinic), a public health organization (e.g., government agencies such as, for example, the U.S. Centers for Disease Control (CDC)) and combinations thereof. In some embodiments, the generated report may be provided to the requestor and/or any other desired recipient. Example types of requestors described above may also be recipients of a report.

In another aspect, the disclosure provides a method for generating a nucleic acid complex comprising a target nucleic acid molecule. The method comprises, in a reaction mixture, amplifying the target nucleic acid molecule with a forward primer and a reverse primer, under conditions that yield the nucleic acid complex. The nucleic acid complex can comprise an amplified target nucleic acid molecule, where the amplified target nucleic acid molecule comprises a first strand and a second strand that is at least partially complementary to the first strand. The first strand can be coupled to a first particle at a 5′ end of the first strand and the second strand can be coupled to the second particle at a 5′ end of the second strand.

In some embodiments, the forward primer may be linked to the first particle and the reverse primer may be linked to the second particle. The forward and reverse primers may be linked to the first particle and second particle, respectively, including via direct attachment or indirect attachment as described elsewhere herein. Direct and indirect attachments between primers and particles may be via covalent bonds, non-covalent interactions and combinations thereof also as described elsewhere herein.

In some embodiments, the reaction mixture may also comprise the first particle and the second particle and any other reagents necessary for amplification of the target nucleic acid molecule as described elsewhere herein. Moreover, the reaction mixture can comprise additional particles coupled to additional forward and/or reverse primers to yield nucleic acid complexes comprising more than two particles coupled to additional amplified target nucleic acid molecules as described elsewhere herein. For example, in some embodiments, the method can further comprise amplifying the target nucleic acid molecule with a third particle linked thereto the forward primer or the reverse primer to yield the nucleic acid complex. In such embodiments, the nucleic acid complex can further comprise the third particle coupled to the first particle and/or the second particle via an additional amplified target nucleic acid molecule.

Additionally, in some embodiments, the method further comprises detecting the nucleic acid complex. Detection of the nucleic acid complex may be achieved via any suitable mode, including example modes of detection (e.g., optical (e.g., with the aid of an optically-responsive species), spectroscopic, electrochemical, electrostatic, physical, electrical, etc.) described elsewhere herein. In some embodiments, the first and second particle may be metallic particles and detection of the nucleic acid complex may be effected by visual examination.

In some embodiments, the reaction mixture may be contained in a partition. Any suitable type of partition may be used to contain the reaction mixture including example type of partitions described elsewhere herein. In embodiments where the reaction mixture is contained in a partition, the method may further comprise releasing the nucleic acid complex from the partition and detecting the released nucleic acid complex. In some embodiments, the method further comprises isolating the nucleic acid complex, which isolation may occur before, during or following any detection of the nucleic acid complex. Any suitable mode of isolation of the nucleic acid complex may be used including example modes of isolation (e.g., centrifugation, magnetic separation, chromatography, capillary action, chromatography, filtration, sedimentation, affinity capture (e.g., affinity capture on a support), etc.) described elsewhere herein.

In another aspect, the disclosure provides a method for generating a nucleic acid complex comprising a target nucleic acid molecule. The method comprises, in a reaction mixture, amplifying the target nucleic acid molecule with a forward primer and a reverse primer to yield an amplified target nucleic acid molecule. The forward primer and the reverse primer can each comprise a 3′ end capable of being extended in a primer extension reaction and a hairpin structure. Additionally, the amplified target nucleic acid molecule can comprise a first strand comprising a first overhang sequence at one end of the amplified target nucleic acid molecule and a second strand comprising a second overhang sequence at the other end of the amplified target nucleic acid molecule. Moreover, the method further comprises contacting the amplified target nucleic acid molecule with a first particle and a second particle to yield a nucleic acid complex. The nucleic acid complex can comprise the amplified target nucleic acid molecule complexed with the first particle and the second particle. The amplified target nucleic acid molecule may be complexed with the first particle via sequence complementarity between the first overhang sequence and a first capture sequence linked to the first particle and complexed with the second particle via sequence complementarity between the second overhang sequence and a second capture sequence linked to the second particle.

In some embodiments, the method further comprises ligating the first strand to the second capture sequence and/or ligating the second strand to the first capture sequence. Ligation may be achieved via any suitable method including via the action of a ligase enzyme present in the reaction mixture such as, for example, a DNA ligase. In addition, the reaction mixture may comprise one or more reagents necessary for amplification of the target nucleic acid molecule, such reagents necessary for nucleic acid amplification described elsewhere herein.

In some embodiments, the method further comprises isolating and/or detecting the nucleic acid complex. Isolation and/or detection of the nucleic acid complex may be completed using any suitable modes of isolation and detection, including example modes of isolation and detection described elsewhere herein.

As used herein, an “overhang sequence” generally refers to a single-stranded sequence associated with the 5′ or 3′ end of a strand of a double-stranded nucleic acid molecule. An overhang sequence may be useful for linking (e.g., via hybridization via sequence complementarity) a double-stranded nucleic acid molecule with another nucleic acid molecule.

Moreover, in some embodiments, the forward primer and/or the reverse primer may comprise a spacer region which cannot be copied via a primer extension reaction. The spacer region of a primer may be useful in generating an overhang sequence on an amplified target nucleic molecule that is derived from the primer. The overhang sequence can be generated because any primer sequence 5′ of a spacer region may also not be copied due to the presence of the spacer region preventing further action of a polymerase during a primer extension reaction. A spacer region may comprise a single nucleotide, a series of nucleotides, and/or a non-nucleic acid species. Non-limiting examples of species that can be included in a spacer region include a polyethylene glycol (PEG), 1′,2′-Dideoxyribose (an abasic site), a carbonaceous linker (e.g., a multi-carbon linker, a C3 spacer, Spacer, Spacer 9, Spacer 18 available from Integrated DNA Technologies) and combinations thereof.

An example of a forward or reverse primer having a 3′ end capable of being extended in a primer extension reaction, having a hairpin structure and a spacer region is schematically depicted in FIG. 3A. As shown in FIG. 3A, a primer 300 is configured to comprise a hairpin structure and comprises a 3′ end that is capable of being extended in a primer extension reaction. In the loop region 301 of the hairpin structure, the sequence of the primer 300 comprises a spacer region 302 that cannot be copied via a primer extension reaction. The primer also comprises a stem region 303. Moreover, a linear configuration of the primer shown in FIG. 3A is schematically depicted in FIG. 3B. As shown in FIG. 3B, the primer comprise an overhang sequence region 304 that is 5′ of the spacer region and a priming sequence region 305 that is 3′ of the spacer region 302. In the hairpin configuration shown in FIG. 3A, a portion of the overhang sequence region 304 is hybridized with a portion of the priming sequence region 305, which forms the stem region 303.

The melting temperature (T_(m,1)) of the overhang sequence 304 when hybridized with a target nucleic acid sequence on a nucleic acid molecule may be less than the melting temperature (T_(m,2)) of the portion of the overhang sequence region 304 hybridized with the portion of the priming sequence region 305 in the stem region 303, which melting temperatures may both be less than the melting temperature (T_(m,3)) of the priming sequence 305 when hybridized with its target nucleic acid sequence on a template nucleic acid molecule. When exposed to a temperature that is greater than T_(m,2), the primer 300 shown in FIG. 3A can adopt the linear configuration shown in FIG. 3B. Moreover, where such a temperature is also less than T_(m,3), the priming sequence 305 can prime a target nucleic acid sequence because the priming sequence 305 does not melt from the target nucleic acid sequence at temperatures less than T_(m,3). For the example primer 300 shown in FIG. 3A and FIG. 3B, T_(m,1) is 28° C., T_(m,2) is 55° C. and T_(m,3) is 59° C.

An example of the functionality of primer 300 shown in FIG. 3A and FIG. 3B is schematically depicted in FIG. 3C. As shown in FIG. 3C, primer 300 is provided in a reaction mixture and upon exposure 310 to the appropriate temperature (T) (e.g., T_(m,1) and T_(m,2)<T<T_(m,3)), primer 300 linearizes. The linearized primer 300 can then prime 320 a nucleic acid molecule 306 via priming sequence region 305 and the nucleic acid sequence for the priming region 305 on nucleic acid molecule 306 that is also included in the reaction mixture. The priming sequence 305 can be extended 307 at its 3′ end in a primer extension reaction (e.g., via the action of a polymerase). Moreover, nucleic acid molecule 306 is not extended to include a sequence complementary to overhang sequence 304 in the primer extension reaction (or a subsequent primer extension reaction) due to the presence of the spacer region 302. The primer extension reaction yields a double-stranded nucleic acid molecule comprising primer 300, nucleic acid molecule 306 linked to single-stranded overhang sequence 304.

The reaction mixture may also include a particle 308 that is coupled to a capture sequence 309 at the 3′ end of the capture sequence. The capture sequence 309 may comprise a sequence that is at least partially complementary to the overhang sequence 304. In such cases, overhang sequence 304 can hybridize 330 with capture sequence 309 such that the double-stranded molecule linked to overhang sequence 304 is coupled to particle 308. Following hybridization of overhang sequence 304 and capture sequence 309, the 3′ end of nucleic acid molecule 306 of the double-stranded nucleic acid molecule can be ligated to the capture sequence 309 at its 5′ end such that the double-stranded molecule is covalently immobilized to the particle 308.

An example method of amplifying a double-stranded nucleic acid molecule (e.g., a target nucleic acid molecule) with a primer similar to primer 300 shown in FIG. 3A/FIG. 3B and generating a nucleic acid complex is schematically depicted in FIG. 4. As shown in FIG. 4, a double stranded nucleic-acid molecule 401, a forward primer 402, a reverse primer 403 and other reagents (e.g., a polymerase, dNTPs, co-factors, etc.) necessary for amplification of double-stranded nucleic acid molecule 401 may be provided in a reaction mixture. The forward primer 402 and the reverse primer 403 are configured to comprise a hairpin structure and a 3′ end that can be extended in a primer extension reaction, similar to the example primer 300 depicted in FIG. 3A and FIG. 3B.

The reaction mixture may then be subject to conditions 410 suitable for amplification of the double-stranded nucleic acid molecule 401 via forward primer 402 and reverse primer 403 to generate a plurality of amplified double-stranded nucleic acid molecules 404. For example, the temperature of the reaction mixture may be cycled such that the double-stranded nucleic acid molecule 401 is amplified via forward primer 402 and reverse primer 403. One strand of an individual amplified double-stranded nucleic acid molecule may comprise a first overhang sequence 405 at its 5′ end and the other strand of the individual amplified double-stranded nucleic acid molecule may comprise a second overhang sequence 406 at its 5′ end.

The amplified double-stranded nucleic acid molecules 404 may then be contacted with one or more first particles 406 and one or more second particles 407. In some cases, the reaction mixture may initially comprise the first particles 406 and the second particles 407 prior to amplification of the double-stranded nucleic acid molecule 401. In other cases, the first particles 406 and the second particles 407 may be added to the reaction during or after amplification of the double-stranded nucleic acid molecule 401. Additionally, an individual first particle of first particles 406 comprises one or more copies of a first capture sequence 408 that is coupled to the individual first particle at its 3′ end. The first capture sequence 408 is at least partially complementary to the overhang sequences 405 of the amplified double-stranded nucleic acid molecules 404. Moreover, an individual second particle of second particles 407 comprises one or more copies of a second capture sequence 409 that is coupled to the individual second particle at its 3′ end. The second capture sequence 409 is at least partially complementary to the overhang sequences 406 of the amplified double-stranded nucleic acid molecules 404.

Upon contact with the first particles 406 and the second particles 407, overhang sequences 405 and 406 can hybridize with capture sequences 408 and 409, respectively via sequence complementarity such that an individual amplified double-stranded nucleic acid molecule 404 is coupled to an individual first particle 406 and/or an individual second particle 407. As the overhang sequences are positioned at the 5′ ends of each strand of an individual amplified double-stranded nucleic acid molecule 404, each amplified double-stranded nucleic acid molecule 404 is coupled to an individual first particle 406 and individual second particle 407 at the 5′ end of one of its component strands. In cases where one or both of the first particles 406 and second particles 407 comprise a plurality of capture sequences 408 and 409, respectively, multiple amplified double-stranded nucleic acid molecules 404 can couple to each particle such that a nucleic acid complex 411 can be generated. Following hybridization of capture sequences and overhang sequences, the 3′ ends of each strand of each amplified double-stranded nucleic acid molecule can be ligated (e.g., via the action of a ligase) to its adjacent capture sequence 408 or 409 such that the double-stranded molecule is covalently attached to the respective capture sequence. Where appropriate, the nucleic acid complex 411 and/or the amplified double-stranded nucleic acid molecules of the nucleic acid complex 411 can then be isolated and/or detected as described elsewhere herein.

In another aspect, the disclosure provides a method for nucleic acid amplification. The method comprises annealing a forward primer linked to a first particle to a nucleic acid strand and annealing a reverse primer linked to a second particle to a complement strand of the nucleic acid strand. Next, the method further comprises extending the forward primer and the reverse primer in a template-directed manner to yield a first double-stranded nucleic acid molecule linked to the first particle and a second double-stranded nucleic acid molecule linked to the second particle. Additionally, the method further comprises denaturing the first double-stranded nucleic acid molecule and the second double-stranded nucleic acid molecule to generate a first single-stranded molecule linked to the first particle and a second single-stranded molecule linked to the second particle. Furthermore, the method further comprises annealing the forward primer to the second single-stranded molecule and annealing the reverse primer to the first single-stranded molecule to yield a nucleic acid complex comprising an amplified double-stranded nucleic acid molecule. The amplified double-stranded nucleic acid molecule can be linked at one end to the first particle and linked at its other end to the second particle.

The steps of the method of may be performed in a reaction mixture that comprises the first particle linked to the forward primer, the second particle linked to the second primer, the nucleic acid strand and one or more reagents necessary for nucleic acid amplification. Moreover, such a reaction mixture may also comprise additional particles (e.g., a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth or greater particle) linked to the forward and/or reverse primers such that nucleic acid complexes comprising additional particles linked to additional amplified double-stranded nucleic acid molecules are generated. For example, in some embodiments, the method can further comprise amplifying the nucleic acid strand with a third particle linked thereto the forward primer or the reverse primer to yield the nucleic acid complex, where the nucleic acid further comprises the third particle coupled to the first particle and/or the second particle via an additional amplified double-stranded nucleic acid molecule.

In some embodiments, one or more steps of the method may be performed in a partition. In some embodiments, the entire method can be performed in a partition. One or more steps of the method or the entire method may be performed in any suitable type of partition including example types of partitions described elsewhere herein. Additionally, one or more steps of the method (up to all steps of the method) may be repeated for one or more cycles, whereby nucleic acid complexes comprising greater than two particles and/or a plurality of nucleic acid complexes can be generated.

In some embodiments, the method further comprises isolating and/or detecting the nucleic acid complex. Isolation and/or detection of the nucleic acid complex may be completed using any suitable modes of isolation and/or detection including example modes of isolation and detection (e.g., optical detection, spectroscopic detection, physical detection, electrical detection, electrochemical detection, electrostatic detection) described elsewhere herein.

An example method of amplifying a double-stranded nucleic acid molecule with forward and reverse primers coupled to particles to yield a nucleic acid complex is schematically depicted in FIG. 5. As shown in FIG. 5, a double stranded nucleic-acid molecule 501; a plurality of first particles 502 that are each coupled (e.g., covalently coupled) to one or more copies of a forward primer 503 at its 5′ end; a plurality of second particles 504 that are each coupled (e.g., covalently coupled) to one or more copies of a reverse primer 505 at its 5′ end; and other reagents (e.g., a polymerase, dNTPs, co-factors, etc.) necessary for amplification of double-stranded nucleic acid molecule 501 may be provided in a reaction mixture. Forward primer 503 exhibits sequence homology with the 5′ end of strand 501 a of double-stranded nucleic acid molecule 501 and is also at least partially complementary to at least a portion of the 3′ end of strand 501 b of double-stranded nucleic acid molecule 501. Reverse primer 504 exhibits sequence homology with the 5′ end of strand 501 b of double-stranded nucleic acid molecule 501 and is also at least partially complementary to at least a portion of the 3′ end of strand 501 a of double-stranded nucleic acid molecule 501. The reaction mixture can be subjected to conditions 510 suitable to denature the double-stranded nucleic acid molecule 501 into its component strands 501 a and 501 b. For example, the reaction mixture may be brought up to a denaturing temperature suitable to denature double-stranded nucleic acid molecule 501.

Following denaturation of double-stranded nucleic acid molecule 501, strands 501 a and 501 b can be contacted with an individual second particle 504 and an individual first particle 502, respectively. The reaction mixture can then be subject to conditions suitable such that an individual copy of forward primer 503 can anneal to its complementary sequence at the 3′ end of strand 501 b and such that an individual copy of reverse primer 505 can anneal with its complementary sequence at the 3′ end of strand 501 a. For example, the temperature of the reaction mixture may be brought to an annealing temperature such that forward primer 503 and reverse primer 504 anneal to their respective sequences on strands 501 b and 501 a.

Following annealing of forward primer 503 and reverse primer 505, the reaction mixture may be brought to conditions 520 such that the 3′ ends of forward primer 503 and reverse primer 505 are extended in a template-directed manner in a primer extension reaction (e.g., via the action of a polymerase). For example, the temperature of the reaction mixture may be brought to an extension temperature at which the primer extension reaction can take place. After extension of forward primer 503 and extension of reverse primer 505, the individual first particle 502 and individual second particle 504 are coupled to amplified double-stranded nucleic acid molecules that comprise component strands that are substantially the same or are the same as strands 501 a and 501 b of double-stranded nucleic acid molecule 501. Individual first particle 502 is coupled to its amplified double-stranded nucleic acid molecule via the 5′ end of a nucleic acid strand that is substantially the same or the same as nucleic acid strand 501 a of double-stranded nucleic acid molecule 501. Individual second particle 504 is coupled to its amplified double-stranded nucleic acid molecule via the 5′ end of a nucleic acid strand that is substantially the same or the same as nucleic acid strand 501 b of double-stranded nucleic acid molecule 501.

The cycle of denaturing, annealing of additional copies of forward primer 503 and reverse primer 505 can be repeated for each amplified double-stranded nucleic acid molecules to generate additional amplified double-stranded nucleic acid molecules by subjecting the reaction mixture to appropriate conditions 530. For example, the temperature of the reaction mixture may be cycled through suitable denaturation, annealing and extension temperatures, as described above. The additional copies of forward primer 503 and reverse primer 505 can be provided by additional individual first particles 502 and individual second particles 504 in the reaction mixture. Copies of forward primer 503 coupled to additional individual first particles 502 and copies of reverse primer 505 coupled to additional individual second particles 504 can anneal to complementary sequences on nucleic acid strands coupled to individual second particles 504 and individual first particles 502, respectively. The primers can then be extended in a subsequent primer extension reaction such that an additional amplified double-stranded nucleic acid molecule is generated that is coupled to both an individual first particle 502 and an individual second particle 504. The process can repeat over a desired number of cycles such that a nucleic acid complex 506 is generated via additional copies of forward primer 503 and reverse primer 504 coupled to first particles 502 and second particles 504, respectively, in the reaction mixture. Where appropriate, the nucleic acid complex 506 and/or the double-stranded nucleic acid molecules of the nucleic acid complex 506 can then be detected as described elsewhere herein.

In various aspects described herein, a method may further comprise isolating a nucleic acid complex. For example, a nucleic acid complex may be isolated from a reaction mixture after an amplification reaction. In some embodiments, a nucleic acid complex may be isolated from a reaction mixture prior to, during, or after detecting the nucleic acid complex. Any suitable mode of isolating a nucleic acid complex may be used. Non-limiting examples of modes of isolating a nucleic acid complex include centrifugation, magnetic separation (e.g., via magnetic properties of a particle or a particle of a nucleic acid complex), chromatography (e.g., size chromatography, affinity chromatography), electrophoresis, capillary action (e.g., capillary action through a solid matrix such as filter paper or a pregnancy test strip), filtration, sedimentation, affinity capture and combinations thereof.

Where affinity capture is used to isolate a nucleic acid complex, one or more particles of a nucleic acid complex may comprise an affinity capture agent that binds to its binding partner, which may, for example, be immobilized on a support. Binding of the affinity capture agent of the nucleic acid complex with its binding pair immobilized to the support can immobilize the nucleic acid complex to the support. For example, one or more particles of a nucleic acid complex may comprise a primer that has not been extended or a capture sequence that has not coupled with another nucleic acid molecule. Such a primer or capture sequence may hybridize with a complementary sequence that may be immobilized to a support, such that hybridization of the primer or capture sequence with the complementary sequence isolates the associated nucleic acid complex by immobilizing the nucleic acid complex to the support. In another example, one or more particles of a nucleic acid complex may comprise one or more streptavidin moieties that bind with respective biotin moieties immobilized to a support. Alternatively, for example, the one or more particles may comprise the biotin and the support may comprise the streptavidin. In either case, binding of the streptavidin and biotin can immobilize the nucleic acid complex to the support.

An example of generating a nucleic acid complex and isolating a nucleic acid complex is schematically depicted in FIG. 6. As shown in FIG. 6, a water-in-oil emulsion 601 is provided in a vessel 602. The water-in-oil emulsion comprises a plurality of droplets in a continuous oil phase 603, where each droplet comprises an aqueous reaction mixture 604 that comprises a plurality of two types of particles 605 and 606. The first type of particle 605 can comprise a forward primer the second type of particle 606 may comprise a reverse primer. Alternatively, the first type of particle 605 and second type of particle 606 may comprise a first and second capture sequence, as described elsewhere herein, capable of hybridizing with an overhang sequence of an amplified nucleic acid molecule. The particles in each droplet are free in the droplet reaction mixtures and not initially associated with a nucleic acid complex. Moreover, positive droplets 607 comprise a template nucleic acid molecule 608, whereas negative droplets 609 do not comprise the template nucleic acid molecule 608. The template nucleic acid molecule may be single-stranded or may be double-stranded.

Positive droplets 607 and negative droplets 609 are subjected to conditions 610 suitable to amplify the template nucleic acid molecule 608 to generate amplified nucleic acid molecules associated with a nucleic acid complex 611. As positive droplets 607 comprise the template nucleic acid molecule, the nucleic acid complex 611 is generated positive droplets 607 following amplification of the template nucleic acid molecule 608. Moreover, no nucleic acid complex 611 is generated in negative droplets 609 because negative droplets 609 do not comprise the template nucleic acid molecule 608 and, thus, no amplification occurs.

The water-in-oil emulsion 601 can then be broken 620 into the continuous oil phase 603 and an aqueous phase 612 that comprises a pool of the aqueous reaction mixtures 604 from the positive droplets 607 and negative droplets 609 of the water-in-oil emulsion 601. The aqueous phase 612 can be separated from the continuous oil phase 603 such as, for example, by pouring the oil off from the aqueous phase via separation funnel or transfer out of the vessel 602 via pipetting. The isolated aqueous phase 612 can then be applied 630 to a surface (e.g., an array, such as a nucleic acid array) 613 immobilized to an affinity capture agent 614 such that the nucleic acid complex 611 in aqueous phase 612 are immobilized to the surface. For example, the affinity capture agent 614 may be a capture sequence that is complementary to a primer or capture sequence associated with one or more particles of the nucleic acid complex 611. In another example, the affinity capture agent 614 may be a member of a binding pair that binds with the other member of the binding pair that is coupled to one or more particles of the nucleic acid complex 611. The isolated complex 611 may then be detected, including via example modes of detection described elsewhere herein.

In various aspects described herein, a method may comprise detecting one or more nucleic acid molecules (e.g., amplified nucleic acid molecules, double-stranded nucleic acid molecules, single-stranded nucleic acid molecules, target nucleic acid molecules, nucleic acid molecules associated with a nucleic acid complex), nucleic acid sequences (e.g., target nucleic acid sequences) and/or nucleic acid complexes. Detection of any of these species may be qualitative and, in some embodiments, quantitative. In some embodiments, the detection of a nucleic acid complex may be used to determine or assay the absence or presence of a nucleic acid molecule (e.g., a target nucleic acid molecule) and/or nucleic acid sequence (e.g., a target nucleic acid sequence) that is component of the nucleic acid complex. For example, the number of nucleic acid complexes detected can be indicative of the copy number of target nucleic acid molecule in a sample that is amplified to generate the nucleic complexes. Detection of a nucleic acid molecule, nucleic acid sequence or nucleic acid complex may be accomplished with any suitable detection method or modality. In some embodiments, a nucleic acid complex may be isolated prior to, during, or after detection. The particular type of detection method used may depend, for example, on the particular species being detected, other species present during detection, whether or not a detectable species is present, the particular type of detectable species to-be-used and/or the particular application.

Non-limiting examples of detection methods include optical detection, electrical detection, physical detection, spectroscopic detection, electrostatic detection and electrochemical detection. Accordingly, a nucleic acid complex, nucleic acid molecule or nucleic acid sequence may be detected by detecting signals (e.g., signals indicative of an optical property, a spectroscopic property, an electrostatic property or an electrochemical property of the nucleic acid molecule, nucleic acid sequence, and/or nucleic acid complex or an associated detectable species) that are indicative of the presence or absence of the nucleic acid molecule, nucleic acid sequence and/or nucleic acid complex. Optical detection methods include, but are not limited to, visual inspection (e.g., detection via the eye, observing an optical property or optical event without the aid of an optical detector), fluorimetry (e.g., fluorescence, fluorescent energy transfer, quenched fluorescence), UV-vis light absorbance and colorimetric detection. Imaging (e.g., microscopy) equipped with an optical mode of detection (e.g., fluorescence microscopy, light microscopy, etc.) can also be used for optical detection. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel based techniques, such as, for example, gel electrophoresis. Such gel techniques may also be useful for isolating a nucleic acid complex as described elsewhere herein. Electrochemical detection methods include, but are not limited to, amperometry. Electrical detection methods include, but are not limited to, detection of conductance or impedance. Physical detection methods include, but are not limited to, detection of particle size, density, sedimentation rate and/or viscosity.

In some embodiments, detection of a nucleic acid molecule, a particle, a set of particles or a nucleic complex may be achieved with fluid flow. A fluid (e.g., solution, reaction mixture) comprising the species to be detected can be flowed past a suitable detector such that the detector detects the species as it flows past the detector. Alternatively, detection may be completed statically in that a fluid comprising the species to be detected is provided to a detector and detection occurs without fluid flow. For example, a species to be detected may be applied to a slide or other surface and imaged using a camera equipped with an appropriate detector.

Detection of a particle, set of particles or a nucleic acid comprising particles may be achieved, at least in part, based on or more physical properties of the particle(s) or nucleic acid complex. In some embodiments detection of one or more particles or a nucleic acid complex comprising particles may be based, at least in part, by the size of a particle, the size particles in a nucleic acid complex or the size of a nucleic acid complex. In some embodiments, detection of one or more particles or a nucleic acid complex comprising particles may be based, at least in part, by the density of a particle, the densities of particles in a nucleic acid complex or the density of a nucleic acid complex. In some embodiments, detection of one or more particles or a nucleic acid complex may be based on sedimentation rate and/or viscosity of the particle(s) or nucleic acid complex. Moreover, detection of one or more particles or a nucleic acid comprising particles may be achieved, at least in part, based on or more electrical properties of the particle(s) or nucleic acid complex.

In some embodiments, detecting a nucleic acid molecule, nucleic acid sequence or nucleic acid complex may be achieved with the aid of a detectable species. A detectable species may be linked or coupled with a nucleic acid molecule, a nucleic acid molecule associated with a nucleic acid complex and/or any other component of a nucleic acid complex, including covalently and non-covalently (e.g., including intercalation of a double-stranded nucleic acid molecule). Moreover, a detectable species may be included in a reaction mixture that is used to generate a nucleic acid complex and/or for nucleic acid amplification, as described elsewhere herein. Non-limiting examples of detectable species include optically-responsive species (e.g., optically-responsive dyes, optically-responsive oligonucleotide probes (e.g., TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, Lion probes, molecular beacons)) and radiolabels (e.g., ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ³⁵S, or ³H).

In some embodiments, a detectable species may be an optically-responsive dye. (e.g., a fluorescent dye) that generates (or fails to generate a signal) when subjected to the appropriate conditions. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polyp yridyls, anthramycin, methylene blue, phenanthridines and acridines, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, Pyronin Y, Blue View, acridine orange, 7-AAD, actinomycin D, LDS751, phycoerythrin, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, umbelliferone, eosin, a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein), erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those including europium and terbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2(and 3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine (ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

In some embodiments, a nucleic acid complex may be detected via an optical change observed upon formation of a nucleic acid complex. For example, as described elsewhere herein, a nucleic acid complex may be generated from a plurality of particles. The particles may be, for example, metallic particles (e.g., metallic nanoparticles) such as, for example, gold nanoparticles or silver nanoparticles. Free particles, prior to the formation of a nucleic acid complex, may exhibit a first color and the nucleic acid complex comprising the same particles may exhibit a second color. The second color may be detected (e.g., with visual examination and/or with a detector) to identify/quantify the nucleic acid complex and a color change (or absence thereof) from the first color to the second color may be used to assay for the presence or absence of the nucleic acid complex. In some embodiments, the nucleic acid complex may be isolated from other species in order to observe any color change. Moreover, other suitable optical properties of a particle may be used for detection of a nucleic acid complex such as, for example, optical signals received from a semiconductor particle.

In some embodiments, a nucleic acid complex may comprise at least two different types of particles (e.g., particles having two different material compositions, such as, for example gold particles and silver particles). Optical detection of the nucleic acid complex can occur via energy transfer between the two types of particles. For example, a first type of particle can have a wavelength 1a and emission wavelength 1e. A second type of particle can have absorption wavelength 2a and emission wavelength 2e. Emission wavelength 2e can be detected when a solution comprising the nucleic acid complex is excited with wavelength 1a when a nucleic acid complex is present. In the absence of a nucleic acid complex, the first particle and second particle are separate and emission wavelength 2e is not detected when the particles are excited with wavelength 1a.

An example of generating a nucleic acid complex via nucleic acid amplification, isolating the generated nucleic acid complex and detecting the nucleic acid complex is schematically depicted in FIG. 1. Two reaction mixtures 101 and 102 are provided. Both reaction mixtures comprise a first type of particle 103 that comprises a forward primer and a second type of particle 104 that comprises a reverse primer. Alternatively, particle 103 and particle 104 may comprise a first and second capture sequence, as described elsewhere herein, capable of hybridizing with an overhang sequence of an amplified nucleic acid molecule. Particles 103 and 104 are free in the reaction mixtures and not initially associated with a nucleic acid complex. In some embodiments, particles 103 and 104 are both metallic particles, such as, for example gold or silver nanoparticles. Reaction mixture 101 comprises a template nucleic acid molecule 105 that can be amplified in the presence of particles 104 and 105 such that a nucleic acid complex is generated comprising one or more amplified nucleic acid molecules and particles 104 and 105. Reaction mixture 102 does not comprise the template nucleic acid molecule 105. The template nucleic acid molecule may be single-stranded or may be double-stranded. Moreover, the color of both reaction mixtures is red due to the color of particles 103 and 104.

Reaction mixtures 101 and 102 are subjected to conditions 106 suitable to amplify the template nucleic acid molecule 105 to generate amplified nucleic acid molecules associated with a nucleic acid complex 107. As reaction mixture 101 comprises the template nucleic acid molecule, the nucleic acid complex 107 is generated in reaction mixture 101 following amplification of the template nucleic acid molecule 105. Moreover, no nucleic acid complex 107 is generated in reaction mixture 102 because reaction mixture 102 does not comprise the template nucleic acid molecule 105 and, thus, no amplification occurs. Upon formation of the nucleic acid complex 107, the color of reaction mixture 101 shifts from a red color to a blue color due to the coupling of particles 104 and 105 in nucleic acid complex 107. The color of reaction mixture 102 remains red, since no nucleic acid complex is generated.

The blue color of reaction mixture 101 can be detected with visual examination to determine that reaction mixture 101 comprises nucleic acid complex 107 (and, thus, amplified nucleic acid molecules). The red color of reaction mixture 102 can be detected with visual examination to determined that reaction mixture 102 does not comprise nucleic acid complex 107 (and, thus, does not comprise an amplified nucleic acid molecule). In cases where isolation of the nucleic acid complex 107 is helpful to observe its blue color (e.g., due to, for example, possible interference from other species in reaction mixture 101), the nucleic acid 107 may be isolated 108 (e.g., via chromatography or filtration) from reaction mixture 101.

The presence 109 of a blue band is indicative of the blue color of the nucleic acid complex 107 in the reaction mixture (and, thus, template nucleic acid molecule 105 in the reaction mixture). The absence 110 of a blue band is indicative that reaction mixture 102 does not comprise nucleic acid complex 107 (and, thus, does not comprise nucleic acid molecule 105). The blue color of the nucleic acid complex 107 is a result of particles 104 and 105 aggregating (via amplification) to generate nucleic acid complex 107.

An example of isolating and detecting a nucleic acid complex is schematically depicted in FIG. 2. As shown, a reaction mixture 201 is contained in a vessel 202 and comprises a nucleic acid complex. The vessel 202 is enclosed in a sealed vessel 203. A sealed vessel 203 can be used to prevent contamination of the reaction mixture 201. Vessel 203 also comprises a solid matrix 204 (e.g., a piece of filter paper or pregnancy test strip) and a mechanism 205 (e.g., a puncture mechanism) capable of generating an outlet in the bottom of vessel 202. The outlet is generated 206 such that reaction mixture 201 is applied 207 to solid matrix 204. Via flow due to capillary action 210, the contents of reaction mixture 201 are separated. Due to its larger size compared to other reagents in reaction mixture 201 (e.g., free particles, reagents for primer extension reactions, primers, etc.), the movement of the nucleic acid complex in reaction mixture 201 is retarded compared to the other reagents. Thus, a band 208 can be generated on the solid matrix 204 that corresponds to the nucleic acid complex and is separate from a band 209 that is generated further down the solid matrix and represents other reagents in the reaction mixture 201. The separation of the bands 208 and 209 can be detected upon visual examination (where possible) or may be detected with a suitable type of detector (e.g., an optical detector). Moreover, the absence of band 208 indicates that reaction mixture 201 does not comprise a nucleic acid complex.

Various aspects described herein may be useful in conducting multiplex nucleic acid amplification reactions. Multiplex nucleic acid amplifications may be useful in a variety of different applications with non-limiting examples that include detection and quantification of small percentage gene copy number differences with precision; detection and quantification of sequence mutations (e.g., insertions, deletions) and rare-mutations (e.g., rare mutations in low-prevalence targets associated with cancer); detection and quantification of pathogens (e.g., detection and absolute quantification of bacterial and viral loads, detection and quantification of low-level pathogens that contaminate food and water supplies); generation of references and standards (e.g., generation of absolute reference standards for genetic measurements, metrology, and cross-laboratory measurements); preparation of sequencing libraries; quantification of sequencing libraries with or without reference standards (e.g., absolute quantification of sequencing libraries and validation of sequencing results without reference standards); detection and quantification of gene expression changes (e.g., detection of gene express changes for absolute transcript quantification without a reference gene); detection and quantification of genetically modified organisms (GMOs) (e.g., detection and absolute quantification of a foreign gene(s) in plants); detection and quantification of circulating tumor cells (CTCs) or other rare nucleic acids that may be present in the blood; analysis of gene copy number in a sample; analysis of pathogen loads and pathogen load changes in a biological sample; analysis of transcription level in response to drug treatment; environmental testing; forensic identification; clinical diagnostics and detection of a nucleotide polymorphism or genotype, such as HLA or HMC typing.

In some embodiments, compositions and methods described herein may be useful for RNA analysis. In such analysis, RNA can be reverse transcribed via the action of a reverse transcriptase to generate cDNA. The cDNA can then be amplified to generate a nucleic acid complex using particles or a set of particles described elsewhere herein. The nucleic acid complex, once formed, can then be isolated and/or detected using any suitable methods, including those described elsewhere herein.

An example method of multiplex nucleic acid amplification is schematically depicted in FIG. 7. As shown in FIG. 7, a nucleic acid sample 701 (e.g., a sample comprising DNA) that is suspected to comprise or comprises one or more target nucleic acid sequences is provided in a vessel 702. An aliquot of the nucleic acid sample 701 can be provided to each of a plurality of vessels 703. In the example shown in FIG. 7, the nucleic acid sample is aliquoted into four vessels, however aliquots of the nucleic acid sample 701 may be provided to any suitable number of vessels. In each vessel, a primer set (e.g., a primer set comprising at least a forward primer and a reverse primer) is provided that is configured to amplify a nucleic acid molecule that comprises a unique target nucleic acid sequence. For example, a forward primer may be provided that is at least partially complementary to a target nucleic acid sequence and a reverse primer may be provided that is at least partially complementary to a complement of the target nucleic acid sequence.

In the example shown in FIG. 7, each primer set is indicated by “A”, “B”, “C” and “D” and each set is configured to amplify a nucleic acid molecule that comprises one of four unique target nucleic acid sequences. Moreover, the primers in each primer set may be provided as coupled to a particle (e.g., coupling via the 5′ end of a primer) as described elsewhere herein or may be provided free from a particle as described elsewhere herein. In cases where the primer is provided free from a particle, the primer may be configured in a hairpin structure and the hairpin structure may also include an overhang sequence that is unique to the particular priming sequence of the particular primer (and, thus, the particular target nucleic acid sequence). In addition to the primer sets, additional reagents necessary for amplifying a nucleic acid molecule (e.g., a polymerase, dNTPs, suitable buffers, co-factors) and generating a nucleic acid complex (e.g., particles coupled to capture sequences complementary to any overhang sequences, etc.) are also provided to the vessels 703 to generate a pre-reaction mixture 704 in each vessel.

Reagents suitable for generating a water-in-oil emulsion (e.g., oil, a surfactant, etc.) can then be added 710 to each pre-reaction mixture 704 such that a water-in-oil emulsion 705 comprising a plurality of aqueous droplets in a continuous oil phase is generated in each vessel. The aqueous droplets in each water-in-oil emulsion 705 comprise an aqueous reaction mixture that includes the contents of the corresponding pre-reaction mixture 704. In some embodiments, a water-in-oil emulsion 705 may be generated such that some of the aqueous droplets in the water-in-oil emulsion 705 do not comprise a nucleic acid molecule to be amplified. Such control may be exerted, for example, by controlling the level of dilution of the sample in the pre-reaction mixtures 704 and/or the amount of reagents added 710 to generate the water-in-oil emulsion 705.

The droplets from each water-in-oil emulsion 705 can then be pooled 720 into a common water-in-oil emulsion 706 provided to common vessel 707. The common water-in-oil emulsion 706 (or water-in-oil emulsions 703) may then be subject to conditions 730 suitable for amplifying nucleic acid molecules in each droplet (e.g., via the primer set present in each droplet) and generating a nucleic acid complex during or after amplification as described elsewhere herein. For example, the temperature of the common water-in-oil emulsion 706 may be cycled as described elsewhere herein. As a result of amplification, nucleic acid complexes may be generated 740 (e.g., corresponding to the closed circles in 740) in droplets that comprise a particular primer set and one or more nucleic acid molecules that comprise the corresponding target nucleic acid sequence. In a droplet where no nucleic acid molecule is present or no nucleic acid molecule that comprises the target nucleic acid sequence corresponding to the primer set in the droplet is present, a nucleic acid complex may not be generated (e.g., corresponding to the open circles in 740) in the droplet due to the lack of the target nucleic acid sequence.

The common water-in-oil emulsion 706 can then be broken 750 such that a two-phase mixture comprising the oil 708 from the continuous oil phase of the common water-in-oil emulsion 706 and a pooled aqueous mixture 709 that comprises the aqueous reaction mixtures of the droplets of the common water-in-oil emulsion 706. The pooled aqueous mixture 709 can be separated from the oil 708 and provided 760 to an array 711. Each position of the array 711 can comprise a surface immobilized thereto one or more capture sequences that are at least partially complementary to a free primer or capture sequence associated with a particular target nucleic acid sequence (and, thus, nucleic acid complex). The positions of the array can be arranged such that a particular column, row or other configuration of positions corresponds to a particular capture sequence or set of capture sequences and, thus, a particular target nucleic acid sequence.

In the example shown in FIG. 7, each column of the array 711 corresponds to a particular primer set (e.g., “A”, “B”, “C” and “D” described above) corresponding to a particular target nucleic acid sequence and is labeled “A′”, “B′”, “C′” and “D′”. Via the capture sequences immobilized to the array 711, the corresponding free primer(s) or capture sequence(s) of the nucleic acid complexes that are generated can bind to their appropriate positions on the array 711. As each free primer or capture sequence of a particular nucleic acid complex is unique to a particular target nucleic acid sequence, the nucleic acid complex can be identified as corresponding to the particular target nucleic acid sequence based on which site(s) it binds to on the array 711. In the example shown in FIG. 7, a closed circle at an array position represents binding of a nucleic acid complex and an open circle at an array position represents no binding of a nucleic acid complex at the array position. Binding of a nucleic acid complex can be detected via any suitable mode, including modes of detection described elsewhere herein. The positions that comprise a particular bound nucleic acid complex (and, thus, a particular target nucleic acid sequence) can be counted 770 (e.g., digitally counted) and the counting data used for a downstream application.

An additional example of multiplex nucleic acid amplification and its possible use in nucleic acid sequencing is schematically depicted in FIG. 8. As shown in FIG. 8, a nucleic acid sample 801 (e.g., a sample comprising DNA) that is suspected to comprise or comprises one or more target nucleic acid sequences is provided in a vessel 802. An aliquot of the nucleic acid sample 801 can be provided to each of a plurality of vessels 803. In the example shown in FIG. 8, the nucleic acid sample is aliquoted into four vessels, however aliquots of the nucleic acid sample 801 may be provided to any suitable number of vessels. In each vessel, a primer set (e.g., a primer set comprising at least a forward primer and a reverse primer) is provided that is configured to amplify a nucleic acid molecule that comprises a unique target nucleic acid sequence. For example, a forward primer may be provided that is at least partially complementary to a target nucleic acid sequence and a reverse primer may be provided that is at least partially complementary to a complement of the target nucleic acid sequence.

In the example shown in FIG. 8, each primer set is indicated by “A”, “B”, “C” and “D” and each set is configured to amplify a nucleic acid molecule that comprises one of four unique target nucleic acid sequences. Moreover, the primers in each primer set may be provided free from a particle as described elsewhere herein. In cases where the primer is provided free from a particle, the primer may be configured in a hairpin structure (e.g., as in the example shown in FIG. 3 and FIG. 4) and the hairpin structure may also include an overhang sequence that is unique to the particular priming sequence of the particular primer (and, thus, the particular target nucleic acid sequence). In addition to the primer sets, additional reagents necessary for amplifying a nucleic acid molecule (e.g., a polymerase, dNTPs, suitable buffers, co-factors) and generating a nucleic acid complex (e.g., particles coupled to capture sequences complementary to any overhang sequences) are also provided to the vessels 803 to generate a pre-reaction mixture 804 in each vessel. In some embodiments, particles provided with capture sequences may also comprise a common affinity capture agent, such as a member of binding-pair (e.g., biotin).

Reagents suitable for generating a water-in-oil emulsion (e.g., oil, a surfactant, etc.) can then be added 810 to each pre-reaction mixture 804 such that a water-in-oil emulsion 805 comprising a plurality of aqueous droplets in a continuous oil phase is generated in each vessel. The aqueous droplets in each water-in-oil emulsion 805 comprise an aqueous reaction mixture that includes the contents of the corresponding pre-reaction mixture 804. In some embodiments, a water-in-oil emulsion 805 may be generated such that some of the aqueous droplets in the water-in-oil emulsion 805 do not comprise a nucleic acid molecule to be amplified. Such control may be exerted, for example, by controlling the level of dilution of the sample in the pre-reaction mixtures 804 and/or the amount of reagents added 810 to generate the water-in-oil emulsion 805.

The droplets from each water-in-oil emulsion 805 can then be pooled 820 into a common water-in-oil emulsion 806 provided to common vessel 807. The common water-in-oil emulsion 806 (or water-in-oil emulsions 803) may then be subject to conditions 830 suitable for amplifying nucleic acid molecules in each droplet (e.g., via the primer set present in each droplet) and generating a nucleic acid complex during or after amplification as described elsewhere herein. For example, the temperature of the common water-in-oil emulsion 806 may be cycled as described elsewhere herein. As a result of amplification, nucleic acid complexes may be generated 840 (e.g., corresponding to the closed circles in 840) in droplets that comprise a particular primer set and one or more nucleic acid molecules that comprise the corresponding target nucleic acid sequence. In a droplet where no nucleic acid molecule is present or no nucleic acid molecule that comprises the target nucleic acid sequence corresponding to the primer set in the droplet is present, a nucleic acid complex may not be generated (e.g., corresponding to the open circles in 840) in the droplet due to the lack of the target nucleic acid sequence. The nucleic acid complexes that are generated may be structurally similar to the example nucleic acid complex 411 shown in FIG. 4, in that the amplified double-stranded nucleic acid molecules of the generated nucleic acid complex can be coupled to the particles of the generated nucleic acid complex via hybridization between capture sequences of the particles and overhang sequences of the amplified double-stranded nucleic acid molecules.

The common water-in-oil emulsion 806 can then be broken 850 such that a two-phase mixture comprising the oil 808 from the continuous oil phase of the common water-in-oil emulsion 806 and a pooled aqueous mixture 809 that comprises the aqueous reaction mixtures of the droplets of the common water-in-oil emulsion 806. The pooled aqueous mixture 809 can be separated from the oil 808, if desired. Additionally, the pooled aqueous mixture 809 can be subject to conditions suitable to ligate (e.g., via the addition of a ligase to the pooled aqueous mixture 809 or a ligase already present in the pooled aqueous mixture 809) 860 amplified double-stranded nucleic acid molecules associated with nucleic acid complexes to respective capture sequences associated with the amplified double-stranded nucleic acid molecules. Following ligation of amplified double-stranded nucleic acid molecules to capture sequences, nucleic acid complexes can be denatured 870 into component particles comprising single strands of amplified double-stranded nucleic acid molecules, such that particles and single-strands are no longer associated in a nucleic acid complex.

Component particles and strands can then be provided 880 to a sequencing array 811. Each position of the sequencing array 811 can comprise a surface immobilized thereto one or more affinity capture agents that can bind with a nucleic acid complex. In some embodiments, for example, the one or more affinity capture agents may comprise capture sequences that are at least partially complementary to a free primer or capture sequence associated with a particular target nucleic acid sequence or even the target nucleic acid sequence itself. The positions of the array can be arranged such that a particular column, row or other configuration of positions corresponds to a particular capture sequence or set of capture sequences and, thus, a particular target nucleic acid sequence. In cases where the component particles comprise a common affinity capture agent (e.g., biotin, an affinity capture agent may comprise an agent (e.g., streptavidin) capable of binding the common affinity capture agent (e.g., biotin).

Via the affinity capture agents immobilized to the sequencing array 811, the corresponding binding species (e.g., free primer(s), capture sequence(s), a common affinity capture agent or a sequence of the single-stranded nucleic acid molecule coupled to a particle) of the component particles can bind to positions on the sequencing array 811. In the example shown in FIG. 8, a closed circle at an array position represents binding of particles and an open circle at an array position represents no binding of particles at the array position. The single-stranded nucleic acid molecules coupled to the component particles can then serve as templates in a nucleic acid sequencing reaction 890, such as, for example, a sequencing-by-synthesis sequencing reaction. Sequences obtained from the sequence reaction can be detected via any suitable mode, including example modes of detection described elsewhere herein.

An example of processing a nucleic acid complex for a sequencing reaction is shown in FIG. 9. As shown in FIG. 9, a nucleic acid complex 901 generated during nucleic acid amplification may comprise a plurality of particles 902 coupled to amplified double-stranded nucleic acid molecules 903 via hybridization of capture sequences coupled to the particles (e.g., at the 3′ ends of the capture sequences) with overhang sequences coupled to the amplified double-stranded nucleic acid molecules 903 (e.g., at the 5′ends of the component strands of the amplified double-stranded nucleic acid molecules). The nucleic acid complex 901 may be then subject to conditions suitable to ligate 910 the amplified double-stranded nucleic acid molecules 903 to respective capture sequences.

Following ligation, the nucleic acid complex may be denatured 920 (e.g., at a denaturing temperature and/or with the addition of a denaturing agent (e.g., an alkaline agent)) into component particles 904. Denaturation can separate amplified double-stranded nucleic acid molecules 903 into component particles 904 by separating the strands of amplified double-stranded nucleic acid molecules 903 into component single-stranded nucleic acid molecules 905. As shown in FIG. 9, example component particles 904 are separated and coupled to single-stranded nucleic acid molecules 905 at a 3′ end of the single-stranded molecules 905.

The component particles 904 can then be provided to a surface 906 that is associated with one or more affinity capture agents 907 and 908. In the example shown in FIG. 9, surface 906 comprises capture sequences 907 and 908 that are at least partially complementary to a free capture sequence on the component particles 904 or other sequences on the component particles 904. The capture sequences 907 and 908 can bind the component particles 904 via hybridization with the appropriate species coupled to the component particles 904. In some embodiments, the component particles 904 may comprise a common member of a binding pair (e.g., biotin) and the affinity capture agent of the surface 906 may comprise the other member of the binding pair (e.g., streptavidin). The two members of the binding pair can bind, such that the component particles 904 bind with the surface 906.

As shown in FIG. 9, binding immobilizes the single-stranded nucleic acid molecules 905 that are associated with the component particles to the surface 906. The single-stranded molecules 905 can serve as templates in a sequencing reaction, such as, for example a sequencing-by-synthesis reaction. Detection of the sequencing reaction can be completed via any suitable mode of detection, including example modes of detection described elsewhere herein.

In various aspects described herein, a method may comprise amplification of a nucleic acid molecule. In general, nucleic acid amplification may occur in a reaction mixture in which the nucleic acid molecule to be amplified is provided along with additional reagents (e.g., forward primers, reverse primers, polymerases, dNTPs, co-factors, suitable buffers, etc.) necessary for amplification of the nucleic acid molecule. The reaction mixture may then be subjected to conditions (e.g., appropriate temperatures, addition/removal of heat, buffer concentrations, etc.) suitable for amplifying the nucleic acid molecule.

For example, a single or double-stranded nucleic acid molecule may be provided in a reaction mixture that also comprises additional reagents (e.g., a forward primers and reverse primers described elsewhere herein, a polymerase, dNTPs, co-factors, buffers, other enzymes (e.g., a reverse transcriptase to generate cDNA from RNA, a ligase, etc.) necessary for amplification of the single or double-stranded nucleic acid molecule. In some embodiments, the temperature of the reaction mixture may be cycled repeatedly through a denaturation temperature (e.g., to denature, separate or melt double-stranded nucleic acid molecules into component nucleic acid strands), an annealing temperature (e.g., to anneal or hybridize a primer to each of the component nucleic acid strands) and an extension temperature (e.g., to extend or add nucleotides to the annealed primers in a primer extension reaction via the action of a polymerase) in order to amplify the single-stranded or double-stranded nucleic acid molecule. The cycling of the temperature of a reaction mixture may be achieved, for example, with the aid of any suitable thermocycler instrument or other type of heating device. In some embodiments, denaturation of a double-stranded nucleic acid molecule may be achieved via a denaturing agent, such as, for example an alkaline agent (e.g. sodium hydroxide (NaOH)). In some cases, amplification of a nucleic acid may be achieved isothermally such as, for example, without a change in temperature of a reaction mixture.

A nucleic acid amplification reaction can include the use and action of a polymerase. During a primer extension reaction, a polymerase can generally add, in template-directed fashion, nucleotides to the 3′ end of a primer annealed to a single-stranded nucleic acid molecule. Any suitable polymerase may be used for a primer extension reaction, including commercially available polymerases. Non-limiting examples of polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, Phusion DNA polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerase with 3′ to 5′ exonuclease activity, KleTaq DNA polymerase, and variants, modified products and derivatives thereof.

In some embodiments, a suitable denaturation temperature may be, for example, about 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C. or higher. In some embodiments, a suitable annealing temperature may be, for example, about 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., or higher. In some embodiments, a suitable extension temperature may be, for example, about 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., or higher. In some embodiments, annealing and denaturation steps may be combined such that they occur at the same temperature.

Any suitable type of nucleic acid amplification reaction may be used to amplify a nucleic acid molecule. One example of a nucleic acid amplification reaction is a polymerase chain reaction (PCR) that relies on repeated cycles of primer annealing, primer extension and denaturing of amplified nucleic acid molecules as described above. Additional non-limiting examples of types of nucleic acid amplification reactions include reverse transcription, ligase chain reaction, nested amplification, multiplex amplification, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, multiple displacement amplification (MDA); and variants of PCR that include real-time PCR, hot start PCR, inverse PCR, methylation-specific PCR, allele-specific PCR, assembly PCR, asymmetric PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, thermal asymmetric interlaced PCR, and touchdown PCR. In some embodiments, digital PCR and other amplification processes can be used in conjunction with any of the amplification methods, particles, primers and/or capture sequences described herein.

Additional aspects of the disclosure provide systems for assaying or identifying the presence of a target nucleic acid molecule/target nucleic acid sequence in a sample and/or executing methods of the disclosure. In one aspect, the disclosure provides a system for analyzing the content(s) of a solution. The system can comprise a detection cell that is adapted to contain or direct a solution containing a nucleic acid complex that comprises a double-stranded nucleic acid molecule linked to at least a first particle and a second particle. The double-stranded nucleic molecule may comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule. In addition, the system may also comprise a detector that is linked to the detection cell and detects signals indicative of the presence or absence of the nucleic acid complex in the solution. Moreover, the system may also comprise a computer processor that is linked to the detector and programmed to receive signals (e.g., signals are indicative of the presence of absence of the nucleic acid complex) from the detector and determine if the nucleic acid complex is present or absent in the solution based on the detected signals.

In some embodiments the detection cell may comprise a vessel (e.g., an example type of vessel described elsewhere herein), a well (e.g., a microwell, a machine microwell, micro-fabricated micro-wells or cavities), an array of wells (e.g., a microwell plate, a molded microwell), and/or a support (e.g., an example type of support described elsewhere herein). In some embodiments, the detection cell may comprise a fluid flow path, such as one or more channels of a microfluidic device. In such embodiments, the solution may be provided to and/or directed within the detection cell and/or other system component via fluid flow. In some embodiments, a system may also comprise one or more pumps that can be configured to flow the solution to and/or through the detection cell and any other system components. In some embodiments, the one or more pumps may be fluidically connected to the detection cell and/or the detector in order to provide and/or the solution to the detection cell. Detection of nucleic acid complexes can occur as nucleic acid complexes flow past the detector in the fluid flow path. In some embodiments, the system may comprise one or more reagent reservoirs, where and individual reagent reservoir can contain the solution and/or any other reagents for the detection of the nucleic acid complex (e.g., a detectable species) prior to its containment in the solution and/or reagents. The reagent reservoirs may be fluidically connected to the detection cell via a fluid flow path, such as one or more channels of a microfluidic device.

In some embodiments, the detection cell may comprise the solution. Moreover, the solution may or may not be contained within a partition. Where the solution is contained in a partition, the partition may be any suitable type of partition, including example types of partitions (e.g., droplets of an emulsion, wells, cavities) described elsewhere herein. In some embodiments, the nucleic acid complex may comprise a plurality of double-stranded nucleic acid molecules linked to greater than two particles as described elsewhere herein. In some embodiments, the solution may comprise a plurality of nucleic acid complexes. The detector can be configured to detect the plurality of nucleic acid complexes and the computer processor can be programmed to determine the presence of the plurality of the nucleic acid complexes (and/or any associated nucleic acid molecules associated with the nucleic acid complexes).

Moreover, the detector may be any suitable type of detector, the type of which may depend upon the particular mode of detection utilized. For example, where a system is configured for optical detection, the detector can be an optical detector (e.g., a spectrophotometer, a UV-vis light absorbance spectrophotometer, a fluorimeter, a colorimeter, a camera (e.g., a charge-coupled device (CCD) camera, an electron charge coupled device (EMCCD) camera), photodiode, a photodiode array an etc.). Additional non-limiting examples of detectors include a spectroscopic detector (e.g., a mass spectrometer, a nucleic magnetic resonance (NMR) spectrometer, a particle sizer, an electrical detector, an infrared spectrometer, an electron paramagnetic resonance (EPR) spectrometer) and an electrochemical detector.

Linkages between various system components can depend on the particular system components being linked. The detection cell and the detector may be electronically linked such that the detection cell electronically communicates with the detector. Moreover, a linkage between the detector and a detection cell may depend, for example, on the particular mode of detection. For example, a detector and a detection cell may be optically linked such that light passes through, from and/or to the detection cell to/from the detector. Moreover, the computer processor may be electronically linked to the detector such that the signals detected by the detector are transmitted by the detector and received by the computer processor electronically. In some embodiments, the detection cell may also be linked (e.g., electronically linked) to the computer processor. In addition, the computer processor may also be programmed to control detector operation (e.g., such as timing of detection, detector configuration for particular detection modes or solution, etc.), detection cell operation, or operation of any other component (e.g., pumps). Furthermore, various components of the system may be contained in a housing. In some embodiments, the detector and detection cell may be contained in the same housing or may be contained in separate housings. In some embodiments, the computer processor may be contained in the same housing as the detector and/or detection cell or may be contained in a separate housing. In some embodiments, all components of the system may be included in a single housing.

In some embodiments, the computer processor may be included as part of a computer system. The computer system may be housed separately from the detector and/or detection cell or may be housed together with one or both components. For example, as shown in FIG. 10, the computer processor 1005 (e.g., a central processing unit (CPU)) may be included as part of computer system 1001 and can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1001 can also include memory or memory location 1010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1015 (e.g., hard disk), communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage and/or electronic display adapters. The memory 1010, storage unit 1015, interface 1020 and peripheral devices 1025 (e.g., keyboards, mice, sounds systems, microphones, printers, or other input or output devices) can be in communication with the computer processor 1005 through a communication bus (solid lines), such as a motherboard. The storage unit 1015 can be a data storage unit (or data repository) for storing data. The computer system 1001 can be operatively coupled to a computer network (“network”) 1030 with the aid of the communication interface 1020. The network 1030 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1030, in some embodiments, may be a telecommunication and/or data network. The network 1030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1030, in some embodiments with the aid of the computer system 1001, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1001 to behave as a client or a server.

The computer processor 1005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. Examples of operations performed by the computer processor 1005 can include fetch, decode, execute, and writeback. The storage unit 1015 can store files, such as drivers, libraries and saved programs. The storage unit 1015 can store programs generated by users and recorded sessions, as well as output(s) associated with the programs. The storage unit 1015 can store user data, e.g., user preferences and user programs. The computer system 1001, in some embodiments, can include one or more additional data storage units that are external to the computer system 1001, such as an additional storage unit that is located on a remote server that is in communication with the computer system 1001 through an intranet or the Internet.

The computer system 1001 can communicate with one or more remote computer systems through the network 1030. For instance, the computer system 1001 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1001 via the network 1030.

Methods described herein and instructions for operating the detection cell, detector and any other component of the system (e.g., pumps) can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1005. In some embodiments, the code can be retrieved from the storage unit 1015 and stored on the memory 1010 for ready access by the processor 1005. In some situations, the electronic storage unit 1015 can be precluded, and machine-executable instructions are stored on memory 1010. The code can be pre-compiled and configured for use with a machine have a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

Additional aspects of the disclosure provide kits that may include particle sets, primers, other reagents (e.g., one or more reagents necessary for amplification of a nucleic acid molecule), and instructions suitable for amplifying a nucleic acid molecule, conducting primer extension reactions, assaying for a target nucleic acid molecule and/or generating (and, in some cases, detecting) a nucleic acid complex. In one aspect, the disclosure provides a kit for assaying the presence or absence of a target nucleic acid strand in a sample having or suspected of having the target nucleic acid strand. The kit may comprise a first particle, a second particle, and instructions for using the first and second particles to identify the presence or absence of the target nucleic acid strand in the sample via a primer extension reaction. The first particle may comprise a first primer that has a first nucleic acid sequence that exhibits sequence homology to a portion of the target nucleic acid strand. The second particle may comprise a second primer that has a second nucleic acid sequence that exhibits sequence homology to a portion of a complement nucleic acid strand of the target nucleic acid strand. In addition, the first nucleic acid sequence may be different than the second nucleic acid sequence.

In some embodiments, the first particle and/or the second particle may be contained in a vessel. The first and/or the second particle may be contained in any suitable type of vessel including example types of vessels described elsewhere herein. Moreover, the first particle and/or the second particle may be any suitable type of particle (including example types of particles described elsewhere herein), may comprise any suitable type of material (including example types of particle materials described elsewhere herein), and/or may be of any suitable particle size (including example particle sizes described elsewhere herein). In some embodiments, the first particle and second particle comprise the same materials. In some embodiments, the first particle and second particle may comprise different materials.

In some embodiments, the kit may further comprise one or more reagents suitable for generating a water-in-oil emulsion or oil-in water emulsion. Non-limiting examples of such reagents include an aqueous media (e.g., water, a buffer, etc), an oil (e.g., mineral oil) and a surfactant (e.g., ABIL EM90 (from Evonik Industries)), ABIL WE 90 (from Evonik Industries), Triton-X100, Tween 80, Span 80). In some embodiments, the kit may further comprise a detectable species (e.g., an optically-responsive species) that can permit identification of the target nucleic acid strand and/or an amplified target nucleic acid strand. In some embodiments, the kit may further comprise one or more reagents included in a reaction mixture, such as reagents (e.g., polymerase, nucleotides (e.g., dNTPs), other enzymes, buffers, co-factors, etc.) necessary for performing the primer extension reaction.

EXAMPLES Example 1: Preparation of Polymeric Particles

Provided herein is an example method of generating polymeric particles that may be used to amplify nucleic acid molecules and/or generate nucleic acid complexes as described elsewhere herein.

An aqueous solution containing 8% acrylamide and 5% bis-acrylamide is prepared and degassed with nitrogen gas (N₂) for 10 minutes. Separately, an oil solution containing 10% surfactant Abil We 90 (from Evonik Industries) in mineral oil is prepared and degassed under high vacuum for 10 minutes. 200 μL of the aqueous solution is then mixed with 10 μL of 6% ammonium persulfate (NH₄)₂S₂O₈ and 20 μL of acrylamide-modified oligonucleotides (e.g., DNA oligonucleotides) or acrydite-modified oligonucleotides to generate a modified aqueous solution. Separately, 400 μL of the oil solution is mixed with 4 μL of tetramethylethylenediamine (TEMED) to generate a modified oil solution.

The modified aqueous solution and modified oil solution is then mixed together and immediately vortexed at about 2500 rpm for 2 minutes to emulsify the mixture. The emulsified mixture is then allowed to stand at ambient temperature for about three hours such that particles are generated in the solution.

Next, 10 mL of ethanol is added to the emulsion and the particles vortexed and then centrifuged at 5000 rpm for 6 minutes (min). Following centrifugation, the supernatant is removed from the mixture via, for example, pipetting. Washing of the particles with 10 mL ethanol followed by vortexing and centrifugation at 5000 rpm for 6 min is then repeated for two additional cycles. The particles are then washed with two cycles of 10 mL water followed by vortexing and centrifugation at 5000 rpm for 6 min. The washed beads are then suspended in 20 mL water and larger particles are allowed to settle to the bottom of the mixture via gravity. Smaller particles remain at the top of the mixture. The smaller particles at the top of the solution can then be removed, diluted with water and filtered through a filter or membrane that selects particles of the desired particle size. The selected particles can then be quantified by SYBR Green staining.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

1.-16. (canceled)
 17. A set of particles for nucleic acid amplification, comprising: a first particle comprising a first primer having a 5′ end and a 3′ end, wherein said first primer is linked to said first particle via said 5′ end of said first primer, and wherein said first primer exhibits sequence homology to a target nucleic acid strand at a 5′ end of said target nucleic acid strand; and a second particle comprising a second primer having a 5′ end and a 3′ end, wherein said second primer is linked to said second particle via said 5′ end of said second primer, and wherein said second primer exhibits sequence homology to a complement nucleic acid strand of said target nucleic acid strand at a 5′ end of said complement nucleic acid strand, wherein a sequence of said target nucleic acid strand to which said first primer exhibits homology is separated from a sequence of said target nucleic acid strand to which said second primer exhibits complementarity by at least about 10 nucleotides. 18.-20. (canceled)
 21. The set of particles of claim 17, wherein said first particle and/or said second particle are contained in a partition.
 22. The set of particles of claim 21, wherein said partition is a droplet, a well or a vessel. 23.-25. (canceled)
 26. The set of particles of claim 17, wherein said first particle comprises at least two of said first primer or two of said second primer.
 27. (canceled)
 28. (canceled)
 29. The set of particles of claim 17, wherein said first and/or second particle comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof.
 30. An isolated nucleic acid complex comprising a double-stranded nucleic acid molecule having at least a first strand and a second strand that is at least partially complementary to said first strand, wherein said first strand is coupled to a first particle at a 5′ end of said first strand, and wherein said second strand is coupled to a separate second particle at a 5′ end of said second strand.
 31. The isolated nucleic acid complex of claim 30, wherein said double-stranded nucleic acid molecule comprises a first end sequence and a second end sequence, wherein said first end sequence is complexed with said first particle via a first capture sequence linked to said first particle, wherein said second end sequence is complexed with said second particle via a second capture sequence linked to said second particle, wherein said first capture sequence is at least partially complementary to said first end sequence and said second capture sequence is at least partially complementary to said second end sequence.
 32. The isolated nucleic acid complex of claim 31, wherein said first capture sequence is linked to said first particle at a 3′ end of said first capture sequence, or wherein said second capture sequence is linked to said second particle at a 3′ end of said second capture sequence.
 33. (canceled)
 34. The isolated nucleic acid complex of claim 30, wherein said first strand is covalently linked to said first particle at said 5′ end of said first strand, or wherein said second strand is covalently linked to said second particle at said 5′ end of said second strand.
 35. (canceled)
 36. The isolated nucleic acid complex of claim 30, wherein said nucleic acid complex is not immobilized to a support. 37.-39. (canceled)
 40. The isolated nucleic acid complex of claim 30, wherein said first particle and said second particle comprise a dimension of about 0.5 nanometers (nm) to about 100 nanometers.
 41. (canceled)
 42. The isolated nucleic acid complex of claim 30, wherein said nucleic acid complex is contained in a partition.
 43. The isolated nucleic acid complex of claim 42, wherein said partition is a droplet, a well or a vessel. 44.-47. (canceled)
 48. The isolated nucleic acid complex of claim 30, wherein said first and/or second particle comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof.
 49. A kit for assaying the presence or absence of a target nucleic acid strand in a sample having or suspected of having said target nucleic acid strand, comprising: a first particle and a second particle, wherein said first particle comprises a first primer having a first nucleic acid sequence that exhibits sequence homology to a portion of a target nucleic acid strand, wherein said second particle comprises a second primer having a second nucleic acid sequence that exhibits sequence homology to a portion of a complement nucleic acid strand of said target nucleic acid strand, wherein said first nucleic acid sequence is different than said second nucleic acid sequence; instructions for using said first and second particles to identify the presence or absence of said target nucleic acid strand in the sample via a primer extension reaction.
 50. The kit of claim 49, wherein said first and second particles are contained in a vessel.
 51. (canceled)
 52. The kit of claim 49, wherein said first and/or second particle comprise a material selected from the group consisting of gold, silver, copper, platinum, palladium, a metal oxide, a polymer, carbon and combinations thereof.
 53. The kit of claim 49, further comprising one or more reagents suitable for generating a water-in-oil emulsion.
 54. (canceled)
 55. The kit of claim 49, further comprising a detectable species that permits the identification of said target nucleic acid strand.
 56. (canceled)
 57. The kit of claim 49, further comprising reagents necessary for performing said primer extension reaction.
 58. The kit of claim 57, wherein said reagents comprise a polymerase and nucleotides. 59.-131. (canceled) 